Nanometer antibody Nb32 targeting herpes simplex virus and application of nanometer antibody Nb32

Abstract

The invention provides a nano antibody Nb32 targeting a herpes simplex virus and application of the nano antibody Nb32. The nano antibody Nb32 has a specific CDR region combined with the extracellular domain of the glycoprotein D on the surface of the HSV, can be combined with the glycoprotein D of the HSV with high affinity, and has a good neutralizing effect on the HSV. The nano antibody provided by the invention can be used for preparing vaccines or medicines for preventing and / or treating herpes simplex virus infection and kits for diagnosing herpes simplex virus infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody engineering and pharmacy, in particular to a nanobody Nb32 targeting herpes simplex virus and a preparation method and application thereof. BACKGROUND

[0002] Herpes simplex virus (HSV) belongs to the alpha subfamily of the herpesviridae family, and has two serotypes, namely herpes simplex virus type 1 (HSV1) and herpes simplex virus type 2 (HSV2). HSV1 mainly causes diseases such as herpes labialis, conjunctivitis, and keratitis. HSV2 mainly causes genital herpes and further increases the risk of HIV infection. The binding of HSV surface glycoprotein gD to downstream receptors is an important link in HSV infection, so gD protein is the main target for the development of neutralizing antibodies.

[0003] There are naturally occurring antibodies without light chains in camelids or sharks, i.e. heavy chain antibodies, whose variable region only consists of heavy chains, which is abbreviated as VHH. The heavy chain variable region of such antibodies is cloned, i.e. a nanobody with a size of only about 13 kDa is obtained. Nanobodies have the advantages of small molecular weight, strong penetration, strong antigen recognition ability, easy engineering, and low production cost. Currently, there is no camel-derived natural nanobody against HSV gD approved for the treatment of herpes virus infection. The wide range and long duration of herpes simplex virus infection easily lead to fatal infection in special groups, and the serious disease burden highlights the urgent need to strengthen clinical intervention for herpes simplex virus infection. Therefore, the development of a nanobody targeting herpes simplex virus and the screening of an HSV therapeutic nanobody with a large drug property are helpful for the treatment of herpes simplex virus infection, reduce the social and economic burden, and have a broad market application prospect. SUMMARY

[0004] To solve the problems in the prior art, one of the purposes of the present application is to provide a nanobody targeting herpes simplex virus.

[0005] The present application adopts the following technical solutions:

[0006] In the present application, a camel is immunized 4 times with the extracellular segment of HSV2 gD expressed by the mammalian expression system HEK 293F, and then peripheral blood lymphocytes (PBMCs) are separated by blood drawing, and total RNA extraction is performed on the separated PBMCs, which is then immediately reverse transcribed into cDNA. The cDNA is used as a template to amplify the nanobody sequence, and finally a nanobody is isolated and obtained, which is named Nb32.

[0007] A Nanobody targeting a Herpes simplex virus, said Nanobody being designated Nb32, said Nb32 having an antigenic complementarity determining region CDR1, CDR2 and CDR3 with an amino acid sequence having a homology of equal to or more than 80%, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0008] The above homologous sequences also comprise an amino acid sequence having one or more, preferably 1, 2 or 3, conservative amino acid mutations, preferably substitutions, insertions or deletions, compared to the sequences as shown in SEQ ID NO: 1-3.

[0009] Preferably, the amino acid sequences of the three antigenic complementarity determining regions CDR1, CDR2 and CDR3 of said Nb32 are as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0010] Preferably, the amino acid sequence of said Nb32 is as follows:

[0011] QVQLVESGGGSVQPGGSLRLSCAAS GYTYSPYLM GWFRQAPGKERE GVAAIYTGGS LPGGSTFYADSVKGRFTISQDKAKNTLYLQMSSLKPEDTAVYYC AANRYFTYGGSMRNPQEYNR WGQGTQVTVSS (SEQ ID NO: 4).

[0012] The above three antigenic complementarity determining regions CDR1, CDR2 and CDR3 of said Nanobody have the following amino acid sequences, respectively, as shown in bold underlined parts above, i.e.:

[0013] CDR1: GYTYSPYLM (SEQ ID NO: 1),

[0014] CDR2: GVAAIYTGGS (SEQ ID NO: 2),

[0015] CDR3: AANRYFTYGGSMRNPQEYNR (SEQ ID NO: 3).

[0016] It is a second object of the present application to provide an antibody targeting a Herpes simplex virus, comprising a Nanobody Nb32 as described above and a Fc domain.

[0017] Preferably, said Fc domain is a human IgG Fc domain, said human IgG Fc domain having an amino acid sequence as shown in SEQ ID NO: 5.

[0018] The fusion of the nanobody Nb32 in the antibody targeting herpes simplex virus and the Fc domain uses a linker common in the art, preferably a flexible linker "GS" linker consisting of glycine and serine residues.

[0019] Preferably, the nanobody or antibody further comprises a detectable label, such as a radioisotope, a luminescent substance, a colored substance, an enzyme or a polyethylene glycol.

[0020] The present application also provides a polynucleotide encoding the nanobody as described above, or encoding the antibody comprising the Fc domain as described above. The nucleotide sequence encoding the Nb32 is shown in SEQ ID NO: 6.

[0021] The present application also provides an expression vector comprising the polynucleotide as described above, and a host cell comprising the expression vector of the polynucleotide.

[0022] Preferably, the host cell is a host cell for expressing exogenous proteins, such as bacteria, yeast, insect cells or mammalian cells.

[0023] The present application provides the use of the nanobody and / or antibody as described above in the preparation of a vaccine or a drug for treating and / or preventing herpes simplex virus.

[0024] The present application also provides the use of the nanobody and / or antibody as described above in the preparation of a kit for diagnosing herpes simplex virus infection.

[0025] The present application finally provides a pharmaceutical composition comprising a pharmaceutically effective dose of the nanobody and / or antibody as described above.

[0026] Preferably, the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.

[0027] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes one or several functions of excipients, stabilizers, antioxidants, colorants, diluents, sustained-release agents; such as starch, lipids, waxes, dextrin, sucrose, lactose, microcrystalline cellulose, gelatin, inorganic salts, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, etc.

[0028] The present application has the following beneficial effects:

[0029] The nanobody (Nb32) is derived from a natural camel heavy chain antibody (VHH), which has the advantages of 1) simple structure, small molecular weight, which is conducive to expression and use; 2) convenient to express in E. coli and various eukaryotic systems; 3) as a single domain antibody, it has only one binding site, which has better permeability, specificity and detection linearity as a diagnostic reagent; 4) it is easy to couple with various fusion proteins or be labeled with various markers; 5) it is easier to prepare a bifunctional antibody, which is more conducive to the development of targeted drugs and cell target directional transport; 6) as a drug development, it has little immunogenicity to humans and is not easy to produce immune rejection. The nanobody provided by the application is expected to be used for herpes simplex virus drug, and has high application value for the prevention, control and treatment of herpes simplex virus. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Fig. 4 is the results of antigen purification and SDS-PAGE analysis, wherein A-B are the results of nickel column purification of the antigen HSV gD, C-D are the results of 24 mL molecular sieve of the antigen HSV gD, and E is the results of SDS-PAGE.

[0031] Figure 2 Fig. 5 is the ELISA results of the monoclonal phage specifically binding to HSV2 gD.

[0032] Figure 3 Fig. 6 is the results of nanobody Nb32-Fc purification and SDS-PAGE analysis, wherein A is the elution chart of nanobody Nb32-Fc purified by Protein A column, and B is the SDS-PAGE results of Nb32-Fc after purification.

[0033] Figure 4 Fig. 7 is the results of analysis of the binding of nanobody Nb32-Fc to HSV gD by ELISA, wherein A is the analysis results of the binding of Nb32-Fc to HSV1 gD; and B is the analysis results of the binding of Nb32-Fc to HSV2 gD.

[0034] Figure 5 Fig. 8 is the results of analysis of the binding of nanobody Nb32-Fc to HSV gD by SPR, wherein A is the analysis results of the binding of Nb32-Fc to HSV1 gD; and B is the analysis results of the binding of Nb32-Fc to HSV2 gD.

[0035] Figure 6 Fig. 9 is the results of HSV neutralization of Nb32-Fc, wherein A is the analysis results of HSV1 neutralization of Nb32-Fc; and B is the analysis results of HSV2 neutralization of Nb32-Fc. DETAILED DESCRIPTION

[0036] For ease of understanding, the technical solutions of the present application will be described in more detail below in conjunction with the embodiments:

[0037] The following embodiments are only used to illustrate the technical solutions of the present application, and are not limiting of the present application; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent replacements and improvements, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

[0038] Example 1: Purification of HSV gD for immunizing camels and screening antibodies

[0039] 1) According to the HSV1 gD nucleotide sequence (SEQ ID NO: 7) and HSV2 gD nucleotide sequence (SEQ ID NO: 8) published by the National Center for Biotechnology Information GenBank database, HSV1 gD and HSV2 gD gene sequences were synthesized by Anhui General Biotechnology Co., Ltd., and were connected to pTT5 vector (referring to Ma H, et al., 2021. Potent Neutralization of SARS-CoV-2 by Hetero-Bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain. J Virol 95: 10.1128 / jvi.02438-20) to construct an expression plasmid, and the expression plasmid was transfected into human embryonic kidney cells HEK 293F cells (referring to Ma H, et al., 2021. Potent Neutralization of SARS-CoV-2 by Hetero-Bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain. J Virol 95: 10.1128 / jvi.02438-20), and cultured at 37°C, 5% CO2, 150 rpm for 4 days, then the supernatant of mammalian cell culture was collected by centrifugation at 3000 rpm for 10 min. HSV1 gD and HSV2 gD were purified by nickel column, eluted with Tris buffer containing high concentration of imidazole, and the obtained HSV1 gD and HSV2 gD were identified by gel filtration chromatography and then analyzed by SDS-PAGE electrophoresis, as shown in Figure 1 Fig. 1, high-purity HSV1 gD and HSV2 gD antigen proteins were obtained.

[0040] 2) Immunize the camels with the high-purity HSV2 gD antigen protein obtained by the foregoing transfection expression purification, and select healthy adult camels for subcutaneous injection. The injection solution is HSV2 gD protein (PBS, pH 7.5) in an equal volume of Freund's adjuvant. The first immunization is at day 0, and Freund's complete adjuvant (Sigma, item number F5881) is used in an equal volume of protein solution. Then, immunization is performed every 14 days, for a total of three times (0.5 mg / time), using Freund's incomplete adjuvant (Sigma, item number F5506) in an equal volume of protein solution. The third time is intramuscular injection. After day 56, booster immunization is performed, and the injection solution is 1 mg of HSV2 gD protein.

[0041] 3) After day 70, the jugular vein blood of the camel is extracted, and peripheral blood lymphocytes are separated using Ficoll cell separation solution (Solarbio, item number P9011).

[0042] 4) Total RNA is extracted using an RNA extraction kit (Omega Bio-tek, item number R6834-01), and genomic DNA is removed. The RNA is reverse transcribed into cDNA using a PrimeScript TM II First-strand cDNA synthesis kit (Takara, item number 6210A).

[0043] 5) Construction of the nanobody phage display library: The coding sequence of the nanobody is obtained by PCR amplification using specific camel VHH primers and the above-mentioned cDNA as the template. The amplified nanobody sequence is inserted into the NcoI and NotI sites of the phagemid pR2 (refer to Ma H, et al., 2021. Potent Neutralization of SARS-CoV-2 by Hetero-Bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain. J Virol 95: 10.1128 / jvi.02438-20) by Gibson assembly. The obtained Gibson assembly product is the initial nanobody phage library, and product recovery is performed.

[0044] 6) Escherichia coli TG1 (MRC Laboratory of Molecular Biology) competent cells are prepared using a 10% glycerol washing method. The activated TG1 is cultured in 300 mL of 2xYT (formula 1L: 16 g of tryptone, 10 g of yeast extract, 5 g of NaCl) medium to an OD 600About 0.6-0.8, after centrifugation at 5000 x g for 15 min, the pellet was washed three times with 250 mL, 250 mL, 100 mL of pre-cooled 10% glycerol, and finally resuspended with 1 mL of 10% glycerol and aliquoted into 500 μL per tube.

[0045] 7) Transformation of E. coli TG1 competent cells using BTX ECM 399 electroporator. Gibson assembled products were mixed with 500 μL of TG1 competent cells and transferred to a 0.1 cm electroporation cuvette, followed by 2.5 KV electroporation. The electroporation product was resuspended in 20 mL of 2xYT medium and incubated at 37 °C, 220 rpm for 1 h. 10 μL of the bacterial solution was diluted into 990 μL of 2xYT, and 40 μL of the bacterial solution was diluted into 160 μL of 2xYT, followed by plating 100 μL and incubation at 37 °C overnight. The next day, the library size was calculated (library size = number of colonies x 10 5 ). The bacteria were spread on 5 150 mm 2xYT plates supplemented with 100 μg / mL ampicillin and 2% glucose to construct the phage library and incubated at 37 °C overnight. Next, the transformed colonies were scraped from the plates, mixed thoroughly with 25% glycerol to a final concentration, snap-frozen in 1 mL aliquots in liquid nitrogen and stored at -80 °C, and the size of the phage library was calculated.

[0046] 8) Amplification of the Nanobody phage display library: To amplify the phage library of Nanobodies, 0.1 ml of the frozen library was thawed on ice, diluted into 100 mL of 2xYT medium supplemented with 100 μg / mL ampicillin and 2% glucose, and incubated at 37 °C, 220 rpm. Next, 1 x 101 12 pfu of KM13 helper phage (MRC Laboratory of Molecular Biology) was added to the culture and incubated at 37 °C (water bath) for 45 min. The cell pellet was separated by high-speed centrifugation and resuspended in 100 mL of 2xYT medium supplemented with 0.1% glucose, 50 μg / mL kanamycin, and 100 μg / mL ampicillin. The cells were incubated at 25 °C, 220 rpm for 20 h to amplify the phage library. After centrifugation, polyethylene glycol (PEG) was added to the culture supernatant to precipitate the phage particles. The precipitated phage particles were resolubilized in 1xPBS (formula: 10 mmol / L Na2HPO4; 1.75 mmol / L KH2PO4; 137 mmol / L NaCl; 2.65 mmol / L KCl; pH 7.2-7.6) and stored in 1 mL aliquots at -80 °C in the presence of 25% glycerol.

[0047] 9) Screening: Purified HSV2 gD was diluted in PBS to a final concentration of 0.1 mg / ml and coated into one well of a 96-well immunoplate ELISA (Nunc maxsorp plates) and one well of the ELISA plate was left out as a negative control. After washing 3 times with 1 x PBS, 260 μL MPBS (1 x PBS containing 5% skimmed milk) was added to each well of the ELISA plate and incubated for 2 hours at room temperature to block unbound sites. Next, the plate was washed 3 times with 1 x PBS and 1 x 10 11 pfu (diluted in 100 μL MPBS) phage library was added to each well and after one hour of incubation at room temperature, washed 15-20 times with PBST (1 x PBS containing 0.1 % Tween 20). Phage displaying specific nanobodies against HSV2 gD were eluted by incubation with trypsin at a final concentration of 0.5 mg / ml for one hour at room temperature. 10 μL of eluted phage was added to 1 mL of E. coli TGI competent cells and incubated at 37 °C (water bath) for 45 minutes to allow for infection, after which the bacterial culture was plated on 2 x YT supplemented with 100 μg / mL ampicillin and 2% glucose at 37 °C overnight.

[0048] 10) Preparation of monoclonal phage: After one round of panning, 96 individual colonies were picked into a 96-well round bottom dish containing 100 μL of 2 x YT medium supplemented with 100 μg / mL ampicillin and 2% glucose (w / v). This was incubated at 37 °C, 220 rpm for 6 hours. Next, 5 μL of this culture was inoculated into a new 96-well round bottom dish containing 100 μL / well of 2 x YT medium supplemented with 100 μg / mL ampicillin and 2% glucose (w / v). The freshly inoculated plate was incubated at 37 °C, 250 rpm for 1.5 hours until the OD 260 was approximately 0.5. 100 μL of 2 x YT / Amp / 2% glucose liquid medium containing 4 x 10 8 pfu of KM13 helper phage was added to each well of the 96 plate and incubated at 37 °C for 45 minutes to allow for infection. After infection, the bacteria were centrifuged at 3500 x g for 15 minutes and the supernatant was discarded, the bacterial pellet was resuspended in 150 μL of 2 x YT / Amp / Kana / 0.1% glucose (w / v) medium and incubated at 25 °C, 220 rpm overnight for approximately 14-16 hours. The next day, the culture was centrifuged at 3500 x g for 30 minutes and 150 μL of supernatant was transferred to a new 96-well plate and stored at 4 °C for screening of the nanobodies.

[0049] 11) Phage ELISA test. 96-well round-bottom immunological ELISA plates were coated with 100 μL of HSV2 gD at a final concentration of 2 μg / mL using lx PBS for dilution and incubated at 4°C for 16 hours. Next, the ELISA plates were washed 3 times with lx PBS. The plates were blocked with MPBS (lx PBS containing 5% skim milk) for 2 hours at room temperature. After completion, the wells were washed 4 times with PBST (PBST). 100 μL of 1 x 10 11 pfu of phage was added to each well and incubated at room temperature for 1 hour. Then the ELSA plates were washed 4 times with PBST. 100 μL of HRP-KM13 diluted 1:8000 in MPBS was added to the ELISA plates per well, incubated at room temperature for 1 hour. Subsequently, the wells were washed 4 times with PBST, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) was added per well, incubated for color development in the dark for 7 minutes, immediately after 50 μL of 1M H2SO4 was added to stop the reaction, and the OD 450nm values were measured by a microplate reader, and the results are shown in Figure 2

[0050] 12) Positive clones with OD 450nm values greater than 1 were picked 95 for sequencing and analyzing the sequence results, and finally Nb32 was determined as a positive phage nanobody specific to HSV gD protein, with an amino acid sequence as shown in SEQ ID NO: 4, and the nanobody Nb32 has a specific CDR region that binds to HSV gD.

[0051] Example 2: Expression and purification of nanobody Nb32-Fc fusion protein

[0052] The peptide gene sequence (SEQ ID NO: 9) for guiding secretion was designed and fused to the N-terminal of the nanobody Nb32 gene to ensure secretion after expression, and human IgG1 Fc was fused to the C-terminal of the nanobody gene through a "GS" flexible linker peptide, and the nanobody gene and human IgG1 Fc were recombined, and then cloned into the mammalian expression vector pTT5 (referring to Ma H, et al., 2021. Potent Neutralization of SARS-CoV-2 by Hetero-Bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain. J Virol 95: 10.1128 / jvi.02438-20).

[0053] ​The constructed pTT5 vector was transfected into human embryonic kidney cells HEK 293F cells (density about 2.5 x 10 6 cells / mL) with polyethyleneimine (PEI) in Freestyle TM 293 expression medium (Invitrogen) at 5% CO2, 150 rpm, 37°C for 4 days, and then the supernatant of mammalian cell culture was collected by centrifugation at 3000 rpm for 15 min. The nanobody-Fc fusion protein was purified by Protein A column and eluted with 0.1 M acetic acid, and the eluted protein was analyzed by SDS-PAGE electrophoresis as shown in Figure 3 FIG. 4, and a high-purity nanobody Nb32-Fc fusion protein was obtained.

[0054] Example 3: Analysis of binding affinity of nanobody to HSV gD

[0055] 1) ELISA analysis of binding affinity of nanobody to HSV gD

[0056] Immuno MaxiSorp plates (Nunc) were coated with 2 μg / mL of HSV1 gD or HSV2 gD protein purified in Example 1 at room temperature for 2 h. After washing the ELISA plates with 1 x PBS 3 times, 260 μL of MPBS was added per well at room temperature for 2 h to block unbound sites. Next, the nanobody Nb32-Fc prepared in Example 2 was gradient-diluted (starting at 300 nM, 4-fold serial dilution, dilution of 11 gradients) with a diluent containing 5% milk in PBST. 100 μL was added per well, and incubated at room temperature for 1 h. After 1 h, the ELISA plates were washed with 1 x PBST 3 times, and HRP-anti-human IgG Fc (Beijing Yiqiao Shenzhou) was diluted with a diluent containing 5% milk in PBST at a ratio of 1:6000 and incubated at room temperature for 1 h. After washing the ELISA plates with 1 x PBST 3 times, 100 μL per well of TMB was added, and reacted at room temperature in the dark for 7 min. Finally, 50 μL of 1 M H2SO4 was added to stop the reaction, and the absorbance at 450 nm was detected.

[0057] The results of the analysis of the ELISA are shown in Figure 4 FIG. 5, and the EC 50 of the nanobody Nb32-Fc to HSV1 gD was 0.1167 nM, and the EC 50 of the nanobody Nb32-Fc to HSV2 gD was 0.1336 nM, indicating that the nanobody Nb32-Fc has a high affinity to HSV gD.

[0058] 2) SPR analysis of binding affinity of Nanobody to HSV gD

[0059] The Nb32-Fc antibody prepared in Example 2 was subjected to affinity determination using a Biacore 8K surface ion resonance analysis system (Cytiva) with HSV1 gD or HSV2 gD. The Nb32-Fc was diluted to 5 pg / mL with sodium acetate at pH 4.0, and the imbolization program was used to couple the Nb32-Fc to the activated Protein A chip, and a control channel without protein coupling was set up. The Nb32-Fc was flowed at a speed of 30 pL / min, and when the coupling value reached 150 Ru, ethanolamine was used for blocking. The HSV gD was diluted 2-fold from 50 nM for 5 gradients, and the sample was added to a 96-well plate. 50 mM NaOH was selected as the regeneration liquid, and the single-cycle kintics / affinity program was used for kinetic determination, with a loading time of 120 s and a dissociation time of 600 s. The results were analyzed using the Biacore evaluation program, as shown in Table 1, the K Figure 5 dissociation constant (K D dissociation constant (K D value of 43.4 pM, indicating that the Nanobody Nb32-Fc has high affinity for HSV gD.

[0060] Example 4: HSV virus titer determination and HSV virus neutralization experiment

[0061] 1) HSV virus titer determination

[0062] The Vero cell line (American Type Culture Collection, item number CCL-81) was selected for the HSV virus (American Type Culture Collection, HSV1 item number VR-1493, HSV2 item number VR-3393) neutralization test, and the long single-layer Vero cells were stably passaged and diluted to a Vero cell suspension of 3.0 x 10 5 The Vero cells were washed twice with PBS, and the HSV virus diluent was added to the Vero cells at 250 pL per well, and incubated at 37°C for 2 h. After the virus liquid was aspirated, 1% methylcellulose 1 ml was added to each plate, and after 72 h, formaldehyde was used for fixation for 30 min, and crystal violet staining solution was added for staining for 15 min. After washing with running water, the number of plaques was counted, and the wells with about 50 plaques were used as the standard. The virus titer calculation formula was: the number of plaques in the well x dilution factor x 4 (pfu / ml). It was detected that the HSV1 virus titer was 1 x 10 8pfu / ml, HSV2 virus titer is 2 x 10 5 pfu / ml

[0063] 2) HSV virus neutralization experiment

[0064] Using the plaque reduction neutralization experiment method, Nb32-Fc antibody is diluted by several times to 7 concentration gradients. In the HSV1 neutralization experiment, the dilution gradient is: 2 μg / mL, 0.33 μg / mL, 0.056 μg / mL, 0.009 μg / mL, 0.0015 μg / mL, 0.0003 μg / mL and 0.0000429 μg / mL. In the HSV2 neutralization experiment, the dilution gradient is: 6.25 μg / mL, 1.5625 μg / mL, 0.390625 μg / mL, 0.0977 μg / mL, 0.0244 μg / mL, 0.0061 μg / mL and 0.00153 μg / mL. Each dilution is repeated three wells, and a virus inoculation antibody-free positive control and a cell negative control are set up. The virus is mixed with the antibody in the same volume, incubated at 37°C for 1 h, shaken twice during the period, and then Vero cells are added and the fixation and staining steps are the same as the previous titer determination method steps.

[0065] As shown in Figure 6 , the results show that the IC 50 of Nb32-Fc antibody to neutralize 50 pfu of HSV1 is 0.0213 μg / mL (0.507 nM) and the IC 50 of Nb32-Fc antibody to neutralize 50 pfu of HSV2 is 0.0211 μg / mL (0.502 nM). The IC 50 values of acyclovir, a classic small molecule drug for treating HSV infection, against HSV-1 and HSV-2 are 0.85 μM and 0.86 μM, respectively (referring to Suzuki M, Okuda T, Shiraki K. Synergistic antiviral activity of acyclovir and vidarabine against herpes simplex virus types 1 and 2 and varicella-zoster virus. Antiviral Res. 2006 Nov;72(2):157-61.). Therefore, the efficiency of Nb32-Fc to neutralize HSV1 is 1676.53 times that of acyclovir, and the efficiency of Nb32-Fc to neutralize HSV2 is 1713.15 times that of acyclovir.

Claims

1. A Nanobody® Nb32 characterized in that, The sequences of the antigenic complementarity determining regions CDR1, CDR2 and CDR3 of the nanobody Nb32 are respectively as shown in any one of the following: 1) an amino acid sequence having a homology of equal to or greater than 80% with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; 2) an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; 3) the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:

3.

2. Nanobody Nb32, characterized in that, The amino acid sequence of the nanobody Nb32 is shown in SEQ ID NO:

4.

3. An antibody targeting a herpes simplex virus, comprising the nanobody Nb32 of claim 1 or 2 and a Fc domain, the Fc domain being a human IgG Fc domain, the amino acid sequence of the human IgG Fc domain being shown in SEQ ID NO:

5.

4. The antibody of claim 3, characterized in that, The antibody further comprises a detectable label, preferably a radioisotope, a luminescent substance, a colored substance, an enzyme or a polyethylene glycol.

5. A polynucleotide encoding the nanobody Nb32 of any one of claims 1 or 2, or encoding the antibody of claim 3 or 4.

6. The polynucleotide of claim 5, the nucleotide sequence of which is shown in SEQ ID NO:

6.

7. An expression vector comprising the polynucleotide of claim 5 or 6.

8. A host cell comprising the expression vector of claim 7.

9. Use of the nanobody Nb32 of claim 1 or 2 or of the antibody of claim 3-4 for the manufacture of a vaccine or a medicament for the treatment and / or prevention of a herpes simplex virus.

10. Use of the nanobody Nb32 of claim 1 or 2 or of the antibody of claim 3-4 for the manufacture of a kit for the diagnosis of a herpes simplex virus infection.

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