CN110204613B - Neutralization antibody for huning virus, preparation method and application thereof - Google Patents
Neutralization antibody for huning virus, preparation method and application thereof Download PDFInfo
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Abstract
The embodiment of the invention provides a neutralization antibody aiming at a huning virus, a preparation method and application thereof, and relates to the technical field of biological medicines.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a neutralization antibody for a huning virus, a preparation method and application thereof.
Background
Junin virus (JUNV) is an important member of the arenavirus genus of the mammalian arenaviridae family, and is a biologically safe quaternary pathogen. It is mainly prevalent in south america and can cause argentine hemorrhagic fever with a fatality rate of about 30% second to ebola, marburg and other virulent viruses. In recent years, along with the rapid circulation of international personnel and materials, JUNV has a tendency to spread to north america, europe, and asia, posing a threat to the global biosafety situation.
Similar to the cognate virus, lassa virus, the JUNV genome contains a large segment L of about 7.2k and a small segment S of about 3.5 k. The L fragment encodes matrix protein Z and RNA polymerase L, while the S fragment encodes the nucleoprotein NP (nucleoprotein) and the envelope glycoprotein GPC (glycoprotin precorsor). The functions performed by the NP protein, the L protein and the Z protein in the life cycle of the virus are gene replication and transcription, budding and composing the capsid matrix protein of the virus respectively. GPC consists of three parts, a Stable Signal Peptide (SSP), an envelope glycoprotein 1(glycoprotein 1, GP1) that mediates viral and receptor adsorption, and an envelope glycoprotein 2(glycoprotein 2, GP2) that mediates membrane fusion. After maturation, GPC of arenavirus is assembled to form a typical GPC trimer structure displayed on the surface of virion, and the GPC structure mediates the adsorption of virus to receptor and the membrane fusion process after adsorption.
The JUNV natural host is a wild mouse, in which symptoms are mild or asymptomatic. Humans are infected by exposure to virus-carrying wild rats or their secretions and excretions, contaminated foods, virus-carrying aerosols, and the like during work and life. Some people (about 30%) gradually become more severe within 1-2 weeks after infection, showing symptoms of persistent fever, dehydration, renal insufficiency, hypotension, severe bleeding at multiple mucosal sites, neurological disorders, shock, and ultimately death from massive hemorrhage and coma.
Although the use of a live virus vaccine (Candid 1) has been approved in the Argentina region to prevent JUNV infection, there are currently no targeted drugs available for clinical treatment of Argentina hemorrhagic fever. Therefore, it is important to develop drugs against JUNV, especially therapeutic antibodies.
Disclosure of Invention
The invention provides a preparation method of a neutralization antibody aiming at a huning virus, which can obtain a horse antiserum product with higher neutralization activity for clinically treating Argentine hemorrhagic fever in a short time and contributes to the emergency treatment of the Argentine hemorrhagic fever.
The invention provides a neutralization antibody aiming at a junin virus, which can effectively inhibit the infection of the junin virus and has higher antiviral activity.
The invention also provides a vaccine, a pharmaceutical composition and an antibody drug conjugate containing the neutralizing antibody, so as to achieve the effect of effectively inhibiting the infection of the Sening virus in a patient.
In addition, the embodiment of the invention also provides application of the neutralizing antibody in preparation of an arenavirus-related medicament and/or reagent, and the application can prepare the arenavirus-related medicament and/or reagent with higher inhibitory activity.
The invention is realized by the following steps:
a preparation method of neutralizing antibody against a virus of junin comprises immunizing animals with the prepared virus antigen of junin to obtain polyclonal antibody, and then specifically purifying polyclonal antibody; the specific purification is a secondary purification of the polyclonal antibody by adopting a huning virus envelope glycoprotein subunit 1 specific affinity chromatography column.
After a series of creative efforts, the inventor of the application finds that the antibody prepared by the secondary purification process has more effective antiviral activity on the huning virus. The specific operation in the secondary purification process is to fix the envelope glycoprotein 1 on activated resin particles to prepare a specific affinity chromatographic column for the envelope glycoprotein 1; the multi-antibody is purified for the second time by using the purification column, and the envelope glycoprotein 1 specific antibody (GP1 specific antibody) with effective neutralization activity is obtained.
Further, the polyclonal antibody may be a polyclonal antibody and/or a polyclonal antibody fragment, and the polyclonal antibody fragment is obtained by concentrating the polyclonal antibody.
The concentration step comprises the steps of adjusting the pH value of a polyclonal antibody solution obtained by immunizing animals with the fenning virus antigen to 2.8-3.2, then adding pepsin for digestion, and concentrating a reaction solution after digestion reaction by adopting a 9-12 kD filter membrane after digestion reaction to obtain a polyclonal antibody fragment.
The polyclonal antibody and the polyclonal antibody fragment are included in the polyclonal antibody, so after secondary purification, the obtained antibodies are GP1 specific antibody and GP1 specific antibody fragment (envelope glycoprotein 1 specific antibody fragment), respectively.
Further, the above-mentioned huining virus antigen is prepared by: constructing a full-length Gene (GPC) of the huning virus envelope glycoprotein to a polyclonal region of an expression vector to obtain an expression plasmid; the expression plasmid is transfected to a host cell for pseudovirus packaging to obtain the huning virus antigen.
Preferably, the resulting antigen against the hunin virus antigen is subjected to antigen purification.
Further, in the present embodiment, the expression vector is preferably pCAGGS, and the pseudovirus is vesicular stomatitis virus pseudovirus VSV Δ G-GFP/G.
Further, in the preparation method, the immunized animal is a horse. The mourning virus antigen obtained above is used for immunizing horses to obtain polyclonal antibodies (horse-derived antibodies). In the application, the preparation and purification process of the equine original antibody has the advantages of short production period and low cost. The murine monoclonal antibody disclosed by the prior art needs to be produced by modification and biological fermentation, and the modified antibody has uncertainty and longer biological fermentation time, so that the preparation process provided by the invention is more mature and is suitable for industrialization of the antibody and the medicine thereof.
The embodiment of the invention also provides a neutralizing antibody against the huningvirus, and the neutralizing antibody is prepared by the preparation method.
The embodiment of the invention also provides a vaccine which is prepared from the neutralizing antibody and/or the neutralizing antibody prepared by the preparation method. Further, the vaccine is against arenaviruses including a junin virus, a machupo virus, a citrullinator virus, a sabina virus and/or a chaparr virus, preferably a junin virus.
The embodiment of the invention also provides a pharmaceutical composition, which comprises the neutralizing antibody and/or the neutralizing antibody prepared by the preparation method. In particular, the pharmaceutical composition is a vaccine against arenaviruses including a junin virus, a machupo virus, a citrullinator virus, a sabina virus and/or a chaparr virus, preferably a junin virus.
The embodiment of the invention also provides an antibody drug conjugate which comprises the neutralizing antibody or the neutralizing antibody prepared by the preparation method. In particular, the pharmaceutical composition is a vaccine against arenaviruses including a junin virus, a machupo virus, a citrullinator virus, a sabina virus and/or a chaparr virus, preferably a junin virus.
In addition, the invention also provides application of the neutralizing antibody in preparing a medicine and/or a reagent related to the arenavirus. Preferably, the arenavirus comprises a junin virus, a machupo virus, a citrullinator virus, a sabina virus and/or a chaparr virus.
The invention has the following beneficial effects:
the embodiment of the invention provides a preparation method of a neutralization antibody aiming at a huning virus, which comprises the steps of immunizing animals with prepared huning virus antigen to obtain a polyclonal antibody, and then carrying out specific purification on the polyclonal antibody, wherein the specific purification is specifically secondary purification on the polyclonal antibody by adopting a huning virus envelope glycoprotein subunit 1 specific affinity chromatography column. The preparation method can prepare the antibody with higher neutralizing activity in a short time, so as to obtain a great amount of horse antiserum products for clinically treating the Argentine hemorrhagic fever, and the preparation method and the products thereof can contribute to the emergency treatment of the Argentine hemorrhagic fever.
In addition, the embodiment of the invention also provides a neutralization antibody, a vaccine, a pharmaceutical composition, an antibody drug conjugate for the huning virus and application of the neutralization antibody in preparation of drugs and/or reagents related to the arenavirus, wherein the neutralization antibody, the vaccine, the pharmaceutical composition and the antibody drug conjugate containing the neutralization antibody have high virus neutralization activity.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a graph showing the results of inhibition of Hunningvirus by group 4 antibodies provided in example 4 of the present invention;
FIG. 2 is a graph showing the results of the antibodies provided in example 3 of the present invention inhibiting Huinin, Maqiupo, Guaranreto, Sabina and Chaparr pseudoviruses;
FIG. 3 is a graph showing the results of inhibition of Hunning pseudovirus by the neutralizing antibody provided in comparative example 1 of the present invention.
Detailed Description
In order to make the objects, 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. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
This example provides a method for producing neutralizing antibodies against the virus junning.
1. Construction of expression vectors
In this example, the full-length Gene (GPC) of the huining virus envelope glycoprotein was obtained by gene synthesis, and the sequence of GPC is shown in SEQ ID No. 1. GPC was constructed into the multiple cloning region of eukaryotic expression vector pCAGGS to obtain expression plasmid pCAGGS-GPC containing GPC.
2. Preparation of a Virus antigen
2.1. Pseudovirus packaging:
and (2) transfecting the constructed expression plasmid pCAGGS-GPC in the step 1 to HEK293T cells (host cells), after 24 hours of expression, infecting the cells by using vesicular stomatitis virus pseudovirus VSV delta G-GFP/G, continuously culturing for 36 hours, and collecting cell culture supernatant to obtain virus liquid, wherein the virus liquid contains a large amount of virus particles.
2.2. Antigen purification:
adding 20% sucrose solution into the bottom of the ultracentrifuge tube, adding the virus solution obtained in step 2.1 into the upper layer, centrifuging at 4 deg.C and 20000rpm/min for 2h, discarding the supernatant, and collecting virus particles (tannin virus antigen) at the bottom.
3. Immunizing animals
Using 1.1% aluminum hydroxide solution as adjuvant, according to 1: volume ratio of 1 adjuvant and the tannin virus antigen obtained in step 2.2 were mixed well to obtain immune mixture, and 5mL of immune mixture was injected into neck and back of horse at multiple spots intradermally, subcutaneously and intramuscularly. Immunizations were performed once every 2 weeks for four times.
4. Preparation of polyclonal antibodies
Plasma was collected 10 days apart after the last immunization in step 3. The collected plasma was diluted with phosphate buffer solution of ph8.0, followed by antibody adsorption on Protein a affinity chromatography column, after washing off non-specific binding proteins, the antibody was eluted with glycine solution of ph3.0, and the eluted antibody (polyclonal antibody) was dialyzed into phosphate buffer solution for use.
5. Preparation of specific antibodies
5.1. The recombinant protein of the hunin virus envelope glycoprotein 1 is fixed on the activated resin particles, and a GP1 specific affinity chromatographic column is prepared. And (3) purifying the polyclonal antibody prepared in the step (4) by using the GP1 specific affinity chromatographic column to obtain a GP1 specific antibody (neutralizing antibody).
5.2. And (3) adjusting the pH value of the solution of the polyclonal antibody obtained in the step (4) to 3.0, adding pepsin to carry out digestion reaction, and after the digestion reaction is ended, concentrating the reaction solution obtained by the digestion reaction by using a 10kD filter membrane to obtain a polyclonal antibody fragment.
The obtained polyclonal antibody fragment is purified for the second time to obtain GP1 specific antibody fragment, and the step of purifying the polyclonal antibody fragment for the second time refers to step 5.1.
Example 2
The activity of the neutralizing antibody obtained in example 1 was verified.
The GP 1-specific antibody or GP 1-specific antibody fragment obtained in example 1 was diluted in serum-free medium with a gradient of 9-15 steps from 200. mu.g/ml, mixed with the pseudovirus hunning and incubated for 1h at 37 ℃.
A mixture of antibody and virus was then added to the Vero-E6 cell surface and infected at 37 ℃ for 1h, with blank and virus as negative and positive controls, respectively. Subsequently, after sufficiently removing the infectious agent, the cells were cultured for 24 hours, and the fluorescence value of each well was quantitatively measured by a luciferase activity assay kit.
The activity of the antibody was evaluated by calculating the inhibition rate of the antibody against viral infection, and by this protocol, the EC50 of the polyclonal antibody, the polyclonal fragment, the GP 1-specific antibody and the GP 1-specific antibody fragment, which inhibit the Sening virus in vitro, were 13.88. mu.g/ml, 7.68. mu.g/ml, 0.76. mu.g/ml and 1.98. mu.g/ml, respectively, were finally obtained.
Example 3
The antibody provided in example 1 is verified to have a broad-spectrum neutralizing effect on highly pathogenic new world arenaviruses.
Antiserum inhibition experiments were performed for Junin virus (JUNV), Machupo virus (Machupo virus, MACV), guanaretto virus (GTOV), Sabia virus (Sabia virus, SABV) and charpah virus (Chapare virus, CHPV), respectively, using the neutralizing antibodies provided in example 1. The polyclonal antiserum obtained in example 1 was diluted with serum-free medium from 1: (20-5120) Dilute 9 gradients, mix with JUNV, MACV, GTOV, SABV or CHPV pseudovirus, respectively, and incubate for 1h at 37 ℃.
A mixture of antisera and virus was then added to the Vero-E6 cell surface and infected at 37 ℃ for 1h, with blank and virus as negative and positive controls, respectively. Subsequently, after sufficiently removing the infectious agent, the cells were cultured for 24 hours, and the fluorescence value of each well was quantitatively measured by a luciferase activity assay kit. The results of antisera inhibiting JUNV, MACV, GTOV, SABV and CHPV pseudoviruses are shown in FIG. 2.
As can be seen from FIG. 2, the Neutralizing Titer (NT) of the neutralizing antibody against JUNV50) 1:2560, neutralizing titers to MACV, GTOV, SABV and CHPV (NT50) Respectively 1 (80-160), 1 (160-320), 1 (320-640) and 1 (160-320).
Example 4
The activity of the antibody provided in example 1 was verified.
The polyclonal antibody, polyclonal antibody fragment polyclonal antibody, GP 1-specific antibody and GP 1-specific antibody fragment prepared in example 1 were used to inhibit the huningvirus, and the operation method of the inhibition experiment is shown in fig. 1 with reference to example 2.
In FIG. 1, IgG is a polyclonal antibody, F (ab')2Being a polyclonal fragment, GP1 specific IgG is a GP1 specific antibody, GP1 specific F (ab')2Is a GP1 specific antibody fragment.
Purification by GP1 specific affinity chromatography from total IgG and F (ab')2GP1 specific IgG and F (ab')2. Specific purification of total IgG and F (ab')2The neutralizing activity of (a) was increased by about 18 times and 4 times, respectively, see fig. 1.
Comparative example 1
Verification the antibody provided in example 1 was compared to antibodies prepared by the prior art.
Experimental methods
Comparative example 1 an antibody was prepared using a DNA (eukaryotic expression vector carrying GPC) antigen, and the other steps were the same as those in example 1. 4 horses were divided into two groups, #1859 and #1863 were pseudovirus-immunized groups (inventive example 1), and #1887 and #1888 were DNA-immunized groups (comparative example 1).
After 4 immunizations, the neutralizing antibody titers in example 1 and comparative example 1 were measured, and the results of the measurements are shown in FIG. 3, in which the data are the results of inhibition of the Hunning pseudovirus. Neutralizing Titer (NT)50): serum dilutions used to inhibit 50% of the virus. The specific detection method is as in example 3.
As shown in FIG. 3, the neutralizing antibody titer of the serum of the pseudovirus immune group provided in example 1 was as high as 1 (2560-5120), while the neutralizing antibody titer of the serum of the DNA group in control example 1 was only 1: 20.
In summary, the embodiment of the present invention provides a method for preparing a neutralizing antibody against a huning virus, the method comprises immunizing an animal with a prepared huning virus antigen to obtain a polyclonal antibody, and then performing specific purification on the polyclonal antibody, wherein the specific purification is specifically a secondary purification of the polyclonal antibody by using a huning virus envelope glycoprotein subunit 1 specific affinity chromatography column. The preparation method can prepare the antibody with higher neutralizing activity in a short time, so as to obtain a great amount of horse antiserum products for clinically treating the Argentine hemorrhagic fever, and the preparation method and the products thereof can contribute to the emergency treatment of the Argentine hemorrhagic fever.
In addition, the embodiment of the invention also provides a neutralization antibody, a vaccine, a pharmaceutical composition, an antibody drug conjugate for the huning virus and application of the neutralization antibody in preparation of drugs and/or reagents related to the arenavirus, wherein the neutralization antibody, the vaccine, the pharmaceutical composition and the antibody drug conjugate containing the neutralization antibody have high virus neutralization activity.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Wuhan Virus institute of Chinese academy of sciences
<120> neutralizing antibody against huning virus, preparation method and application thereof
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1458
<212> DNA
<213> Huning Virus
<400> 1
atgggccagt tcatctcctt catgcaagag atccctacct tcctgcagga ggctctgaac 60
atcgccctgg tggctgtgtc cctcatcgcc attatcaagg gcatcgtcaa cctgtacaag 120
tccggcctgt tccagttctt tgtcttcctg gccctggccg gcaggtcctg taccgaagag 180
gccttcaaga tcggcctgca caccgagttt cagaccgtca gcttttccat ggtcggcctg 240
ttctccaaca atccccacga cctgcccctg ctctgtaccc tcaataagag ccacctctac 300
atcaagggag gcaatgcctc cttccaaatc tccttcgacg atatcgccgt gctcctgccc 360
cagtacgatg tgatcatcca gcatcccgcc gacatgagct ggtgcagcaa gtccgacgac 420
caaatttggc tgagccagtg gttcatgaac gccgtgggac acgattggca cctggaccct 480
cccttcctgt gcaggaacag aaccaagacc gagggcttca tcttccaggt caacacctcc 540
aagaccggcg tgaacgaaaa ctacgccaag aagttcaaga caggcatgca ccacctgtac 600
agggagtatc ccgacagctg ccccaacggc aagctctgcc tgatgaaggc tcagcctaca 660
agctggcccc tgcagtgccc tctggaccat gtgaacaccc tccacttcct gaccaggggc 720
aagaacattc agctgcccag gagatccctg aaggcttttt tctcctggag cctgaccgac 780
agcagcggaa aggacacccc cggaggctac tgcctggagg agtggatgct cgtggctgcc 840
aaaatgaagt gcttcggcaa taccgccgtg gccaagtgca atctcaatca cgattccgaa 900
ttttgcgata tgctgagact cttcgactac aacaagaatg ccattaagac cctgaacgac 960
gagaccaaga aacaggtgaa cctcatgggc cagaccatca atgccctgat tagcgacaac 1020
ctgctcatga agaacaagat tagggagctc atgagcgtgc cctactgcaa ttataccaag 1080
ttctggtacg tgaatcacac actgtccggc cagcactccc tgcccagatg ctggctgatc 1140
aagaacaaca gctacctcaa catttccgac ttcaggaacg actggatcct ggagagcgat 1200
ttcctgatct ccgagatgct gtccaaggaa tactccgaca ggcagggcaa aacccccctg 1260
accctggtgg atatctgctt ctggtccacc gtgttcttta ccgccagcct gttcctgcac 1320
ctcgtgggaa tccccaccca caggcacatt agaggcgagg cctgtcccct gccccacaga 1380
ctgaattccc tgggcggctg tagatgtggc aagtacccca acctgaagaa gcccaccgtg 1440
tggaggagag gccactga 1458
Claims (7)
1. A method for preparing a neutralizing antibody against a virus of Junin, comprising the steps of:
constructing the full-length gene of the huing virus envelope glycoprotein GPC with the sequence shown as SEQ ID No.1 to the polyclonal region of a eukaryotic expression vector pCAGGS to obtain an expression plasmid pCAGGS-GPC containing GPC;
transfecting the constructed expression plasmid pCAGGS-GPC to HEK293T cells, expressing for 24h, infecting the cells by using vesicular stomatitis virus pseudovirus VSV delta G-GFP/G, continuously culturing for 36h, and collecting cell culture supernatant to obtain virus liquid, wherein the virus liquid contains a large amount of virus particles; adding 20% sucrose solution into the bottom of an ultracentrifuge tube, adding virus solution into the upper layer, centrifuging at 4 deg.C and 20000rpm/min for 2h, discarding the supernatant, and collecting bottom virus particles (tannin virus antigen);
immunizing horses with the obtained huning virus antigen, diluting plasma collected after the last immunization with a phosphate buffer solution with pH8.0, then performing antibody adsorption on Protein A affinity chromatography columns, washing off non-specific binding Protein, eluting the antibody with a glycine solution with pH3.0, and dialyzing the eluted antibody (polyclonal antibody) into the phosphate buffer solution for later use; or adjusting the pH value of the obtained multi-antibody solution to 3.0, adding pepsin to carry out digestion reaction, and after the digestion reaction is ended, concentrating the reaction solution obtained by the digestion reaction by adopting a 10kD filter membrane to obtain a multi-antibody fragment;
and then, carrying out secondary purification on the multi-antibody or multi-antibody fragment by using a huning virus envelope glycoprotein 1 GP1 specific affinity chromatographic column to obtain a GP1 specific antibody (neutralizing antibody) or a multi-antibody fragment.
2. A neutralizing antibody against a virus of Hunning, which is produced by the production method according to claim 1.
3. A vaccine comprising the neutralizing antibody of claim 2.
4. A pharmaceutical composition comprising the neutralizing antibody of claim 2.
5. An antibody drug conjugate comprising the neutralizing antibody of claim 2.
6. Use of the neutralizing antibody of claim 2 for the preparation of a medicament related to an arenavirus that is a junin virus, a machupo virus, a citrullinator virus, a sabina virus and/or a copaier virus.
7. Use of a neutralizing antibody according to claim 2 for the preparation of an agent against an arenavirus that is a junin virus, a machupo virus, a citrullinator virus, a sabina virus and/or a copaier virus.
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| WO2014093627A2 (en) * | 2012-12-13 | 2014-06-19 | Children's Medical Center Corporation | Compositions and methods for inhibiting viral entry |
| WO2017214298A1 (en) * | 2016-06-08 | 2017-12-14 | Children's Medical Center Corporation | Compositions and methods for treating arenavirus infection |
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| WO2014093627A2 (en) * | 2012-12-13 | 2014-06-19 | Children's Medical Center Corporation | Compositions and methods for inhibiting viral entry |
| WO2017214298A1 (en) * | 2016-06-08 | 2017-12-14 | Children's Medical Center Corporation | Compositions and methods for treating arenavirus infection |
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