An alpaca-derived nanobody that specifically binds to Chikungunya virus (CHIKV), its preparation method, and its application.

Abstract

This invention belongs to the fields of molecular virology and immunology, and provides an alpaca-derived nanobody that specifically binds to Chikungunya virus (CHIKV), its preparation method, and its applications. The nanobody has a heavy chain variable region (VHH), which comprises the following CDRs: CDR1 (amino acid sequence as shown in SEQ ID NO:1), CDR2 (amino acid sequence as shown in SEQ ID NO:2), and CDR3 (amino acid sequence as shown in SEQ ID NO:3). The VHH includes four frame regions FR1-FR4; FR1, FR2, FR3, and FR4 are arranged alternately with CDR1, CDR2, and CDR3 in sequence. The nanobody of this invention can efficiently neutralize CHIKV virus and inhibit its infection, and has the potential to prepare drugs or kits for the prevention, treatment, and / or detection of CHIKV infection. The nanobody of this invention can bind with high affinity to the E protein of CHIKV, effectively inhibiting CHIKV pseudovirus infection.

Description

Technical Field

[0001] This invention belongs to the fields of molecular virology and immunology, specifically relating to an alpaca-derived nanobody that specifically binds to Chikungunya virus (CHIKV), its preparation method, and its application. Background Technology

[0002] Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes chikungunya fever in humans, characterized by high fever, rash, and severe joint pain. Some patients may develop chronic arthritis, significantly impacting their quality of life. In recent years, CHIKV has caused numerous outbreaks worldwide, particularly in tropical and subtropical regions, becoming a significant public health issue.

[0003] Currently, there are no specific anti-CHIKV drugs on the market, and clinical treatment is mainly symptomatic and supportive. While vaccine development has made some progress, it is not yet widely used. Therefore, developing highly effective and specific anti-CHIKV biological agents is of significant clinical importance. Nanobodies are a novel antibody molecule developed in recent years, derived from heavy-chain antibodies in camelids. They possess advantages such as small molecular weight, high stability, low immunogenicity, and ease of expression and modification. They can recognize antigenic epitopes that are difficult for traditional antibodies to access, making them suitable for multiple fields such as virus neutralization, diagnosis, and treatment. Summary of the Invention

[0004] The purpose of this invention is to provide an alpaca-derived nanobody that specifically binds to Chikungunya virus (CHIKV), its preparation method, and its applications. This nanobody exhibits high neutralizing activity and can effectively inhibit CHIKV infection, demonstrating promising development prospects.

[0005] To achieve the above objectives, the present invention provides the following technical solution: an alpaca-derived nanobody or its antigen-binding fragment that specifically binds to CHIKV, having a heavy chain variable region VHH, wherein the VHH comprises the following CDRs: CDR1 with an amino acid sequence as shown in SEQ ID NO:1, CDR2 with an amino acid sequence as shown in SEQ ID NO:2, and CDR3 with an amino acid sequence as shown in SEQ ID NO:3, wherein the VHH includes four frame regions FR1-FR4; wherein FR1, FR2, FR3 and FR4 are arranged alternately with CDR1, CDR2 and CDR3 in sequence.

[0006] Furthermore, the amino acid sequence of the FR1 frame region is shown in SEQ ID NO:4, the amino acid sequence of the FR2 frame region is shown in SEQ ID NO:5, the amino acid sequence of the FR3 frame region is shown in SEQ ID NO:6, and the amino acid sequence of the FR4 frame region is shown in SEQ ID NO:7.

[0007] The complete amino acid sequence of the heavy chain variable region VHH is shown in SEQ ID NO:8, wherein: positions 1-19 are FR1, positions 29-44 are FR2, positions 52-74 are FR3, positions 100-110 are FR4, CDR1 is inserted between the FR1 frame region and the FR2 frame region, CDR2 is inserted between the FR2 frame region and the FR3 frame region, and CDR3 is inserted between the FR3 frame region and the FR4 frame region.

[0008] The present invention also provides a polynucleotide encoding an alpaca-derived nanobody or antigen-binding fragment thereof that specifically binds to CHIKV as described above, the sequence of which is shown in SEQ ID NO:9.

[0009] The present invention also provides nucleic acid constructs comprising the aforementioned polynucleotides.

[0010] The present invention also provides an expression vector comprising the aforementioned nucleic acid construct.

[0011] The present invention also provides transformed cells comprising the aforementioned polynucleotides, nucleic acid constructs, or expression vectors.

[0012] The present invention also provides pharmaceutical compositions comprising the nanobody or its antigen-binding fragment thereof, and pharmaceutically acceptable carriers and / or excipients.

[0013] The present invention also provides the use of the alpaca-derived nanobody or its antigen-binding fragment that specifically binds to CHIKV in the preparation of kits or medicaments for the prevention, treatment and / or diagnosis of CHIKV infection.

[0014] The present invention also provides nucleic acid constructs comprising the said polynucleotides, expression vectors, and host cells transformed therefrom. The present invention further provides pharmaceutical compositions comprising the said nanobodies and their use in the preparation of kits or medicaments for the prevention, treatment, or diagnosis of CHIKV infection.

[0015] This invention, through immunizing alpacas, constructing a phage display library, and screening for high-affinity nanobodies, yielded nanobodies capable of specifically recognizing CHIKV. Experiments showed that the purified CHIKV E protein has a molecular weight of approximately 95 kDa, while the CHIKV-VHH nanobodies have a molecular weight of 13 kDa and a purity exceeding 95%. Their equilibrium dissociation constant (KD) < 100 nM indicates high affinity. High concentrations (100 nM) and medium concentrations (10 nM) of CHIKV-VHH effectively inhibited pseudovirus infection of 293T cells, while the inhibition rate of 1 nM CHIKV-VHH was relatively lower, approximately 15%. Pseudovirus neutralization activity experiments further demonstrated that CHIKV-VHH, by binding to the CHIKV E protein, further neutralizes CHIKV pseudoviruses, effectively reducing viral load. This antibody effectively neutralizes CHIKV pseudoviruses, exhibiting good binding activity and stability. Attached Figure Description

[0016] Figure 1 The figures show the purification and SDS-PAGE identification results of CHIKVE protein in Example 1 of this invention; in the figures: the left figure is the purification diagram of CHIKVE protein; the right figure is the SDS-PAGE electrophoresis diagram of CHIKVE protein; Figure 2 The image shows the SDS-PAGE image of the purified CHIKVE protein nanobody in Example 2 of this invention; in the image: the left image is the purified CHIKVE protein nanobody; the right image is the SDS-PAGE electrophoresis image of the CHIKVE protein nanobody. Figure 3 This is a kinetic curve of the binding between the nanobody and the CHIKV antigen in Example 3 of the present invention; in the figure: the left figure is the affinity detection curve of the negative control Z6 and the CHIKV E protein; the right figure is the affinity detection curve of the CHIKV E protein nanobody and the CHIKV E protein. Figure 4 The image shows the neutralization detection of CHIKV pseudovirus by nanobody in Example 4 of this invention; in order, they represent negative control, low concentration, medium concentration, and high concentration. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0019] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0021] Example 1: Expression and purification of CHIKV antigen protein: Recombinant p62-E1 protein was prepared using a Bac-to-Bac baculovirus expression system. The coding sequences of CHIKV p62-E1 (GenBank: ABN04188) and ONNV p62-E1 (GenBank: AF079456) were cloned into a modified pFastbac Dual plasmid (Invitrogen Cat#10712024). Green fluorescent protein (GFP) was placed under the P10 promoter for visual expression. The C-terminus of the p62 extracellular domain was linked to the N-terminus of E1 by a 20-residue linking sequence (GGGGS)4 (4 representing 4 GGGGS repeats), thereby bypassing the p62 transmembrane region and the 6K protein. The actual constructs contain amino acids 1-405 of p62 (corresponding to E2 residue 341) and amino acids 1-412 of E1 (i.e., the entire extracellular domain, ending at the E1 transmembrane initiation). Each construct uses its native p62 signal peptide sequence and adds a Twin-Strep-tag II and hexahistine tag to the C-terminus to facilitate purification.

[0022] Recombinant pFastbac Dual plasmid was transformed into DH10Bac™ E. coli, and transfection and viral amplification were performed in sf9 cells. The recombinant protein was expressed in High Five cells for 2 days. The secreted protein is a mature E3-E2-E1 complex, formed by the cleavage of p62 by a furin-like protease. Soluble E protein was purified by nickel affinity chromatography (GE Healthcare) with elution in 20 mM Tris-HCl, 150 mM NaCl, and 15% imidazole (pH 8.0). The purified E protein was then purified by gel filtration in 20 mM Tris-HCl and 150 mM NaCl buffer using a HiLoad 16 / 600 Superdex 200 pg column (GE Healthcare) to obtain highly purified E protein. SDS-PAGE results showed a molecular weight of approximately 95 kDa. Figure 1 As shown.

[0023] Example 2: Alpaca Immunization, Phage Library Construction, and Nanobody Screening and Identification: Purified CHIKVE protein was emulsified with Freund's adjuvant and immunized in alpacas four times. Peripheral blood was collected after the last immunization, PBMCs were isolated, and total RNA (RNeasy® Plus MiniRNA, QIAGEN) was extracted and reverse transcribed to synthesize cDNA (Super Script® Ⅲ First-Strand Synthesis System, Invitrogen). The VHH fragment was amplified twice using nested PCR with the cDNA library. The primer sequences for the two PCRs are shown in Table 1. The corresponding VHH bands were recovered by gel electrophoresis. The obtained VHH fragment and vector were ligated and transformed into E. coli TG1 cells to construct a primary CHIKVE protein nanobody library. The library volume was 1.2 × 10⁻⁶. 9 CFU, insertion rate 100%. The phage library was subjected to three rounds of affinity screening targeting the CHIKV E protein. Single clones were randomly selected for ELISA identification, yielding multiple positive clones. Sequencing confirmed the presence of a nanobody with a unique CDR sequence, named CHIKV-VHH.

[0024] Table 1 Primer Sequences Example 3: Expression and Purification of Nanobodies: The CHIKV-VHH gene was cloned into the pET21a vector, transformed into BL21(DE3) Escherichia coli, and induced to express for 12 h at 16°C with 300 mM IPTG to obtain soluble nanobodies. The target protein was purified using a HisTrap HP 5 mL affinity chromatography column (GE Healthcare). Elution was performed in elution buffer containing 20 mM Tris (pH 8.0), 150 mM NaCl, and 150 mM imidazole. Purification was then performed using a HiLoad 16 / 600 Superdex 75 prep gel filtration chromatography column (GE Healthcare) in buffer containing 20 mM Tris (pH 8.0) and 150 mM NaCl. SDS-PAGE analysis confirmed that CHIKV-VHH with a molecular weight of 13 kDa and a purity of over 95% was obtained. Figure 2 As shown.

[0025] VHH contains the following CDRs: CDR1 with the amino acid sequence shown in SEQ ID NO:1, specifically: TASRFRLDHYVVG; CDR2 with the amino acid sequence shown in SEQ ID NO:2, specifically: IRASAGSTSYADSVK; CDR3 with the amino acid sequence shown in SEQ ID NO:3, specifically: AVDYRYLYSCSLNTSPYAR; VHH includes four frame regions FR1-FR4; FR1, FR2, FR3 and FR4 are arranged alternately with CDR1, CDR2 and CDR3 in sequence.

[0026] The amino acid sequence of the FR1 frame region is shown in SEQ ID NO:4, specifically: DVQLQESGGGLVQPGGSLRLSC; the amino acid sequence of the FR2 frame region is shown in SEQ ID NO:5, specifically: WFRQAPGKEREGVAC; the amino acid sequence of the FR3 frame region is shown in SEQ ID NO:6, specifically: GRFTISEDIAKNTVDLQMHSLKPEDTALYYC; the amino acid sequence of the FR4 frame region is shown in SEQ ID NO:7, specifically: WGQGTQVTVSS.

[0027] The complete amino acid sequence of VHH is shown in SEQ ID NO:8, specifically: DVQLQESGGGLVQPGGSLRLSCTASRFRLDHYVVGWFRQAPGKEREGVACIRASAGSTSYADSVKGRFTISEDIAKNTVDLQMHSLKPEDTALYYCAVDYRYLYSCSLNTSPYARWGQGTQVTVSS.

[0028] Wherein: bits 1-19 are FR1, bits 29-44 are FR2, bits 52-74 are FR3, and bits 100-110 are FR4. CDR1 is inserted between the FR1 and FR2 frame areas, CDR2 is inserted between the FR2 and FR3 frame areas, and CDR3 is inserted between the FR3 and FR4 frame areas. VHH is arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0029] Example 4: Affinity Assay: CHIKV E protein was immobilized on a chip using surface plasmon resonance (SPR) technology. CHIKV-VHH was serially diluted to concentrations of 0.625, 1.25, 2.5, 5, 10, and 20 mM. The affinity of CHIKV-VHH was determined using a Biacore T200 (GE Healthcare) surface plasmon resonance spectrometer. The buffer system was PBST (10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.005% Tween 20). The data were then used to fit the Langmuir binding equation using Biacore™ T200 (GE Healthcare) evaluation software. The results are shown below. Figure 3 As shown, its equilibrium dissociation constant KD < 100 nM, approximately 22.7 Nm, indicating that it has high affinity.

[0030] Example 5: Detection of Neutralizing Activity of Pseudoviruses: The neutralizing activity of nanobodies was detected using the CHIKV pseudovirus system. 293T cells were cultured at 1×10⁻⁶ cells / day. 5 Seeds were seeded in 96-well plates at a density of / mL, and DMEM medium was added at 1×10⁻⁶ mL. −1 1×10 −2 1×10 −3 1×10 −4 1×10 −5 The CHIKV pseudovirus was diluted 100 μL and added to cells. The cells were incubated at 37°C for 72 h. Observation under an inverted fluorescence microscope showed that wells with more than one lesion cell were considered positive. The Reed-Muench method was used to calculate the half-maximal infectious dose (TCID50), which is the viral dilution required to infect half of the cells. The endpoint dilution ratio for TCID50 was 1:1000.

[0031] 293T cells were used at a rate of 1×10 5 The cells were seeded at a density of 1 / mL in 6-well plates, and the structural gene expression plasmid and reporter plasmid were co-transfected into 293T cells using Lipofectamine 2000. The transfected 293T-HPV 16 L1 cells were then seeded at a density of 1×10⁶ cells / mL in 6-well plates. 4The nanobodies were seeded at a density of 1 μM / mL in 96-well plates and serially diluted with DMEM containing 10% fetal bovine serum, ranging from 1 μM to 0.05 nM. The diluted Nb nanobodies and pseudoviruses (1:1000 dilution) were added sequentially to the 96-well plates. COVID-19 RBD antibody 2F2E5 protein and the pseudovirus group served as negative controls. The plates were incubated at 37°C for 1 h. 100 μL of the above mixture was added to the wells of 293T cells, with distilled water as the control group. The plates were incubated at 37°C for 24 h, and then green fluorescence (reporter gene production) was observed under a fluorescence microscope and photographed. Results are as follows: Figure 4 As shown, the results indicated that both high concentrations (100 nM) and medium concentrations (10 nM) of CHIKV-VHH effectively inhibited pseudovirus infection of 293T cells, while the inhibition rate of 1 nM CHIKV-VHH was relatively lower, approximately 15%. Figure 1 As shown in the figure, the results of the pseudovirus neutralization activity experiment further demonstrate that CHIKV-VHH, by binding to the CHIKV E protein, further neutralizes the CHIKV pseudovirus and can effectively reduce viral load.

[0032] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An alpaca-derived nanobody that specifically binds to CHIKV or its antigen-binding fragment, having a heavy chain variable region VHH, characterized in that: The VHH comprises the following CDRs: CDR1 with the amino acid sequence shown in SEQ ID NO:1, CDR2 with the amino acid sequence shown in SEQ ID NO:2, and CDR3 with the amino acid sequence shown in SEQ ID NO:

3. The VHH includes four frame regions FR1-FR4. FR1, FR2, FR3, and FR4 are arranged alternately with CDR1, CDR2, and CDR3 in sequence.

2. The alpaca-derived nanobody or its antigen-binding fragment that specifically binds to CHIKV according to claim 1, characterized in that: The amino acid sequence of the FR1 frame region is shown in SEQ ID NO:4, the amino acid sequence of the FR2 frame region is shown in SEQ ID NO:5, the amino acid sequence of the FR3 frame region is shown in SEQ ID NO:6, and the amino acid sequence of the FR4 frame region is shown in SEQ ID NO:

7.

3. The alpaca-derived nanobody or its antigen-binding fragment that specifically binds to CHIKV according to claim 1, characterized in that: The complete amino acid sequence of the heavy chain variable region VHH is shown in SEQ ID NO:8, wherein: positions 1-19 are FR1, positions 29-44 are FR2, positions 52-74 are FR3, positions 100-110 are FR4, CDR1 is inserted between the FR1 frame region and the FR2 frame region, CDR2 is inserted between the FR2 frame region and the FR3 frame region, and CDR3 is inserted between the FR3 frame region and the FR4 frame region.

4. A polynucleotide encoding an alpaca-derived nanobody or antigen-binding fragment thereof that specifically binds to CHIKV as described in any one of claims 1-3, wherein the sequence of the polynucleotide is shown in SEQ ID NO:

9.

5. A nucleic acid construct comprising the polynucleotide of claim 4.

6. An expression vector comprising the nucleic acid construct of claim 5.

7. Transformed cells comprising the polynucleotide of claim 4, the nucleic acid construct of claim 5, or the expression vector of claim 6.

8. A pharmaceutical composition comprising the nanobody or antigen-binding fragment of any one of claims 1-3, and a pharmaceutically acceptable carrier and / or excipient.

9. Use of the alpaca-derived nanobody or antigen-binding fragment thereof that specifically binds to CHIKV as described in any one of claims 1-3 in the preparation of kits or medicaments for the prevention, treatment and / or diagnosis of CHIKV infection.

10. Use of the pharmaceutical composition of claim 8 in the preparation of a kit or medicament for the prevention, treatment or diagnosis of CHIKV infection.

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