Polypeptide nbm14 that can recognize and neutralize mers-cov and applications thereof

Abstract

The present application relates to a kind of polypeptide NbM14 identifiable and neutralizing MERS-CoV, including 3 complementarity determining regions CDR1-3, sequence as SEQ ID NO:1-3 show.The present application carries out nanobody drug development to MERS-CoV, by preparing MERS-CoV S protein, immunizing bactrian camel, utilizes phage library display nanomantibody platform technology etc., screen to the nanobody VHH specific binding MERS-CoV, identify its CDR sequence, and construct humanized VHH-huFc1;Simultaneously, using pseudovirus neutralization experiment evaluates the curative effect of NbM14 in the treatment of MERS-CoV infection, provides potential detection agent and therapeutic drug for the prevention and treatment of MERS-CoV infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine. More particularly, it relates to a polypeptide capable of binding to MERS-CoV, and to the use of the polypeptide in the preparation of a therapeutic drug and a diagnostic agent for MERS-CoV infection. BACKGROUND

[0002] Middle East Respiratory Syndrome (MERS) was first discovered in a 60-year-old Saudi Arabian male patient who died in June 2012. The incubation period of the disease is 2 to 14 days, and the typical manifestation is acute respiratory infection, with acute onset, high fever (39-40℃), and symptoms such as chills, rigor, cough, chest pain, headache, general muscle and joint pain, fatigue, and loss of appetite. There is no available vaccine and specific treatment method. Although the disease initially occurred in the Middle East region, it gradually spread to 27 countries in Europe, Africa, Asia, and North America with the development of trade, tourism, and other activities. Therefore, the development of safe and effective neutralizing antibodies against the pathogen of MERS has important public health significance in the prevention and control of the epidemic.

[0003] The pathogen causing MERS is MERS-CoV, which belongs to the genus β-CoV and is a single-stranded positive-sense RNA virus with an envelope. The single-stranded RNA genome of MERS-CoV is about 30 kb in size, and contains 10 open reading frames (ORF), encoding 16 non-structural proteins (nsp1-16) and four structural proteins: spike protein (S), envelope protein (E), matrix protein (M), and nucleocapsid protein (N). These four structural proteins form a spherical, crown-like virus particle, with S and N having strong immunogenicity. The S protein is a type I transmembrane glycoprotein in a trimeric state, located on the surface of the virus membrane, mediates virus attachment to host cells and virus-cell membrane fusion, and is the main determinant of cell targeting and pathogenesis. The S protein can induce the body to produce neutralizing antibodies, which play a key role in preventing MERS-CoV infection. Therefore, immunizing animals with S protein and screening antibodies with neutralizing activity from them will be one of the effective strategies for treating MERS-CoV infection.

[0004] In 1993, a new type of natural antibody derived from Camelidae was discovered. The antibody naturally lacks light chains and is composed of heavy chains only, which contain two constant regions (CH2 and CH3), a hinge region and a heavy chain variable region (Variable heavy chain domain, VHH, i.e. antigen binding site), the relative molecular mass of the heavy chain variable region is about 13KDa, which is only 1 / 10 of the conventional antibody, and the molecular height and diameter are in nanometer level, which is the smallest functional antibody fragment available at present, so it is also called Nanobody (Nb). Due to the characteristics of high stability (it will not degrade under the condition of 90℃), high affinity, more than 80% homology with human antibodies, low toxicity and immunogenicity, etc., the Nanobody has been widely used in the research and development of immunodiagnostic kits, imaging, and antibody drugs for tumors, inflammation, infectious diseases and nervous system diseases, etc. SUMMARY

[0005] The present application obtains camel-derived Nanobody by immunizing camel with antigen, which is used for detecting and treating MERS-CoV infection. Based on these studies, the present application provides a polypeptide capable of binding to MERS-CoV, which comprises three complementarity determining regions CDR1-3, and the sequences are shown as SEQ ID NO: 1-3.

[0006] In one embodiment, the polypeptide is a Nanobody.

[0007] In one embodiment, the polypeptide further comprises four framework regions FR1-4, which are staggered in order with the CDR1-3. For example, the FR1-4 sequence can be designed as shown in SEQ ID NO: 4-7 (camel-derived), but the scope of the present application is not limited thereto. It is well known in the art that the specific recognition and binding ability of the antibody is mainly determined by the CDR region sequence, and the FR sequence has little effect, and can be designed according to the species. The FR region sequence of human, mouse or camel origin can be designed to connect the above-mentioned CDR, so as to obtain a Nanobody capable of binding to CD4.

[0008] In one embodiment, the polypeptide is a camel-derived VHH or humanized VHH.

[0009] The present application also provides the use of the above-mentioned polypeptide in the preparation of a detection agent for MERS-CoV or a detection agent for the S protein of MERS-CoV.

[0010] The present application also provides the use of the above-mentioned polypeptide in the preparation of a therapeutic drug for MERS-CoV.

[0011] The present application also provides a nucleic acid encoding the above-mentioned polypeptide.

[0012] The application also provides application of the nucleic acid in preparation of a MERS-CoV treatment drug.

[0013] The application develops a nanobody drug for MERS-CoV, and through preparation of MERS-CoV S protein, immunization of a two-humped camel, and use of a phage library display nanobody platform technology, a nanobody VHH specifically binding to MERS-CoV is screened, the CDR sequence of the nanobody VHH is identified, and a humanized VHH-huFc1 is constructed; meanwhile, the therapeutic effect of NbM14 on MERS-CoV infection is evaluated by using a pseudovirus neutralization experiment, and a potential detection agent and treatment drug for prevention and treatment of MERS-CoV infection are provided. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Antibody titer detection curve of the fourth immunized alpaca against MERS-CoV-S protein after one week.

[0015] Figure 2 Inhibition curve of the fourth immunized camel serum of different dilution against MERS-CoV pseudovirus in vitro infection of ghost cells after one week, with pre-immune serum as a control.

[0016] Figure 3 Screening and identification of MERS-VHH phage antibody library, wherein A is an ELISA detection statistical chart of the phage library after screening against MERS-CoV-S protein; B is an ELISA detection statistical chart of 24, 24 and 40 clones selected from the phage antibody library after the first round (1 st ), the second round (2 nd ) and the third round (3 rd ) screening.

[0017] Figure 4 ELISA detection statistical chart of the prokaryotic expressed VHH antibody, each point represents a clone, and the vertical coordinate is OD450 / OD450 of the blank control against S protein, and a ratio greater than 5.0 is defined as positive.

[0018] Figure 5 SPR detection statistical chart of NbM14 antibody.

[0019] Figure 6 ELISA detection statistical chart of OD450 of NbM14 antibody combined with MERS-S protein at different purification concentrations.

[0020] Figure 7 NbM14 antibody neutralization MERS-CoV pseudovirus infection experiment curve chart. DETAILED DESCRIPTION

[0021] 1. Obtaining of Llama immune and antiserum

[0022] The llama was primed with 250 μg MERS-S protein and 250 μl emulsion mixture of Freund's complete adjuvant, and boosted with MERS-S protein and 250 μl Freund's incomplete adjuvant at 14th day, 28th day and 42th day. After 1 week of the 2nd and 3rd immunization, blood was collected to detect the titer of antiserum. After 1 week of the 4th immunization, 200 ml blood was collected for construction of phage antibody library.

[0023] The titer of antiserum was detected by ELISA. The detection plate was coated with MERS-S-his protein at a concentration of 0.5 μg / ml, 100 μl of gradient-diluted antiserum or purified antibody was added to each well (control: pre-immune llama serum), incubated at 37°C for 1.5 h, washed twice, 1:10000 diluted horseradish peroxidase-labeled Goat anti-Llamma IgG (H+L) secondary antibody was added to each well, incubated at 37°C for 1 h, washed 4-6 times, 100 μl TMB substrate was added, incubated at 37°C for 10 min, 50 μl 0.2M H2SO4 was added to stop the reaction, and OD 450nm was measured. The serum titer detected by ELISA was defined as the highest dilution fold which was more than 2 times of the blank control and greater than 0.2.

[0024] The results are shown in Table 1. Figure 1 As shown in Table 1, the titer of antiserum of 4th immunization was 3.28 x 10 6 Therefore, the antigen can induce llama to produce high-titer antiserum specific to MERS-S protein.

[0025] To further verify whether the high-titer llama antiserum can effectively prevent MERS-CoV virus infection, a virus infection neutralization experiment was performed. Different dilution concentrations of antiserum and pre-immune serum were incubated with MERS-CoV pseudovirus for 60 min, and then transferred to vero cells. After 48 h, the virus load was detected by Glo Max chemiluminescence enzyme labeler (Promega) and the neutralization effect was calculated. The neutralization experiment results showed that the ID90 of MERS-S protein-induced antiserum inhibiting 90% MERS-CoV infection was more than 1000-fold dilution Figure 2 In summary, MERS-S protein induced high-titer antiserum, and the antiserum had high efficiency in inhibiting MERS-CoV pseudovirus infection.

[0026] 2. Construction and screening of VHH phage library

[0027] Peripheral blood of 200 ml from immunized camels was collected, PBMC of camels was obtained by using lymphocyte separation medium (GE Ficoll-Paque Plus), RNA was extracted according to TRIzol manual, and cDNA was reversely transcribed by using oligo (dT), and VHH gene of camels was cloned into phagemid plasmid by primer amplification and molecular cloning technology, TG1 bacteria was transformed to obtain VHH phage library.

[0028] In order to further identify whether the MERS-CoV-VHH phage library is successfully constructed, the VHH target gene of the camel immunized with MERS-S protein is amplified by PCR, and it can be seen that the target band is 450 bp, which is consistent with the expected size, indicating that the MERS-CoV-VHH phage antibody library contains VHH gene. 25 clones are selected for sequencing, and 21 clones have target fragment insertion, and the insertion rate is about 84%. The sequencing results show that the 21 clones have no completely consistent repeated sequences, and the library diversity is 100%. The comparison results show that the difference sequences are mostly in the CDR binding region. It is detected that the CD4-VHH phage antibody library is constructed, and the library capacity is 1.37 x 10 9 .

[0029] With the help of M13KO7 helper phage, the bacteria transformed by VHH-phagemid are used for phage antibody library resuscitation, and PEG / NaCl is used for precipitation. The phage antibody library is coated with 50 μg / ml of MERS-S-His protein for three times. The enriched phage is eluted, transformed, plated, and single clones are selected for phage and CD4 protein ELISA binding identification. The clones with binding value >1.0 are sequenced and cloned into expression vector pcDNA3.1, and transfected 293tt cells to express and produce nanomab.

[0030] The library after panning is combined with MERS-S protein for detection. The phage ELISA results show that the binding value of CD4-VHH phage library before enrichment with CD4 protein is 0.78, and the binding values of phage library after one round, two rounds and three rounds of enrichment are 0.97, 2.59 and 3.34( Figure 3 A). In order to further verify the positive phage rate of MERS-CoV-VHH protein combined in the library after enrichment, 24, 24 and 40 clones are selected from the libraries after the first, second and third rounds of enrichment for single phage ELISA detection. The results show that 50% of the single phage clones in the second round library are positive, and 75% of the phage clones in the third round library are positive, and the Target / Blank ratio is all >5( Figure 3B), the MERS-CoV-VHH phage library with high binding force was successfully enriched by MERS-S protein panning.

[0031] 3. Construction of VHH prokaryotic expression library and VHH expression

[0032] The 2nd-MERS-CoV-VHH and 3rd-MERS-CoV-VHH phage antibody libraries enriched after two rounds and three rounds of panning were subjected to PCR amplification; the gene fragments of all VHHs in the antibody library were obtained and purified, the VHH gene fragments were cloned into a prokaryotic expression vector, transformed into SS320 strain, and a VHH prokaryotic expression antibody library was constructed; the prokaryotic expression antibody library was plated and incubated overnight, and the next day 182 single colonies were randomly selected, the antibody supernatant was induced to express using IPTG, and the antibody supernatant was subjected to ELISA binding detection with S protein.

[0033] The results showed that 49 bacterial supernatants were combined with S protein, and at the same time, not combined with blank control, the read value of S protein binding / read value of blank control was greater than 5.0( Figure 4 ) Among them, the antibody NbM14 was screened out, the sequences of CDR1-3 were as shown in SEQ ID NO: 1-3, and the sequences of FR1-4 were as shown in SEQ ID NO: 4-7.

[0034] 4. VHH-huFc eukaryotic expression

[0035] Through molecular cloning technology, the NbM14 gene was fused with human Fc gene and inserted into pCDNA3.4 eukaryotic expression vector to construct NbM14-huFc-pCDNA3.4 expression plasmid. The constructed NbM14-huFc-pCDNA3.4 was transfected into 293tt cells to express and produce NbM14-huFc (4NB). The cell supernatant was collected for ELISA determination.

[0036] Affinity determination test of antibody NbM14 and MERS-S protein. Fortebio biomolecular interaction platform was used to detect affinity. The antibody was immobilized on Anti-human IgG Fc Capture Biosensors (AHC) probe, the immobilization time was 400 s, then the antigen CD4-his protein was combined, the combination time was 180 s, the dissociation time was 180 s, the antibody-antigen combination and dissociation were observed, the data was derived from the instrument fitting curve. The affinity determination results are shown in Table 1, the affinity of most antibodies can reach 10 -12 (picomole level), and the combination and dissociation curve is shown in Figure 5 It can be seen that we obtained an antibody with high affinity.

[0037] Table 1 4NB334 affinity data

[0038] Clone ID Ka(1 / MS) Kd(1 / S) KD(M) Response(nm) NbM14 9.34E+05 ＜1E-07 ＜1E-12 0.211

[0039] 5. Antibody gradient dilution ELISA

[0040] Coat plates with 0.5 μg / ml MERS-S protein, 100 μl per well, 37°C for 2h, wash 2-4 times, block with 4% BSA, 250 μl per well, 37°C for 1h, wash 2-4 times, add gradient dilution of purified antibody, 100 μl per well, 37°C for 1.5h, wash 2 times, add 1:10000 dilution of horseradish peroxidase labeled anti-human antibody, 100 μl per well, 37°C for 1h, after washing 4-6 times, add 100 μl TMB substrate, 37°C for 10 min, stop reaction with 50 μl 0.2M H2SO4, measure OD450nm. Results are shown in Figure 6 Figure 2, when the antibody NbM14 concentration is as low as 0.00164 μg / ml, the ratio of OD450 of MERS-S protein binding to OD450 of blank control is still greater than 2.

[0041] 5. NbM14 neutralizes MERS-CoV pseudovirus

[0042] MERS-CoV pseudovirus was generated by co-transfecting 293T cells (ATCC) with a plasmid expressing firefly luciferase (pNL43R-E-luciferase) and pcDNA3.1 (Invitrogen) expression vector. Virus supernatant was collected 48h later. Virus titer was determined by luciferase activity in relative light units (Bright-Glo Luciferase Assay Reagent System, Promega Biosciences). Control monoclonal antibody was anti-SFTSV antibody SNB02 (1 mg / ml), and NbM14 was subjected to in vitro neutralization experiment. Antibody was gradient diluted to different concentrations, and incubated with MERS-CoV pseudovirus for 1 hour at 37°C in 5% CO2, then 1x10 4 6. Vero cells were added, and incubated at 37°C in 5% CO2 for 48 hours. The half maximal inhibitory concentration (IC50) of the monoclonal antibody was evaluated by measuring luciferase activity

[0043] Results are shown in Figure 7 Figure 3, NbM14 has good neutralization activity, when the antibody concentration is 0.0407 μg / ml, the inhibition rate can reach 90%.

[0044] From the above experimental results, it can be seen that the antibody NbM14 and the humanized form thereof can specifically recognize and combine with MERS-CoV and S protein thereof, and can neutralize MERS-CoV and block infection thereof, thereby being used for treating MERS.

[0045] The above merely provides the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A polypeptide that binds MERS-CoV, characterized in that, The polypeptide comprises three complementarity-determining regions (CDR1-3), with sequences shown in SEQ ID NO:1-3, and is a nanobody.

2. The polypeptide of claim 1, wherein, It also includes four frame regions FR1-4, which are arranged alternately with CDR1-3 in sequence.

3. The polypeptide of claim 1, wherein, The nanobody is a camel-derived VHH or a humanized VHH.

4. The use of the polypeptide according to any one of claims 1-3 in the preparation of a detection reagent for MERS-CoV or a detection reagent for the S protein of MERS-CoV.

5. The use of the polypeptide according to any one of claims 1-3 in the preparation of a therapeutic agent for MERS-CoV infection.

6. A nucleic acid, characterized in that, The polypeptide is encoded by any one of claims 1-3.

7. The use of the nucleic acid according to claim 6 in the preparation of a therapeutic agent for MERS-CoV infection.

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