Construction and application of infectious clones of RSV-BA9 genotype variants

Abstract

The present invention relates to a kind of RSV-BA9 genotype variant and the construction and application of its attenuated strain infectious clone. By reverse genetics technology, the present invention first constructs and rescues RSV-BA9 genotype variant (comprising an extension of 7 amino acids in the G protein sequence), and then operates by reverse genetics technology to remove the 7 amino acids extended on the variant G protein, and obtains an attenuated strain with decreased viral virulence. The present invention constructs a fast, simple and accurate RSV reverse genetics system through the construction of RSV-BA9 genotype variant and attenuated strain infectious clone and the rescue of two strains. It can be applied to the research on in vitro viral replication mechanism and pathogenic mechanism, neutralizing antibody immune escape, and provides a pathogenic technology platform for the research and development and evaluation of RSV vaccines, antibodies and drugs, etc.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to the construction and application of infectious clones of RSV-BA9 genotype variants. Background Art

[0002] Human respiratory syncytial virus (HRSV) is an RNA virus belonging to the genus Orthopneumovirus in the family Pneumoviridae. High-risk groups for HRSV infection include infants, the elderly, and immunocompromised individuals. HRSV can cause lower respiratory tract infections such as bronchiolitis and pneumonia. Globally, an estimated 33 million children under the age of five suffer from HRSV-associated acute lower respiratory tract infection (ALRI), including approximately 3.6 million hospitalizations and 26,000 deaths among hospitalized children under the age of five.

[0003] RSV has only one serotype, divided into subtypes A and B. The protein with the greatest variation between the two subtypes is the adhesion glycoprotein (G). RSV can be divided into different genotypes by analyzing the nucleotide sequence of the second hypervariable region (HVR2) of the G protein gene. Currently, the dominant genotype of RSV subtype A worldwide is ON1, while the dominant genotype of RSV subtype B is BA9. There have been outbreaks caused by RSV-B BA9. The successful construction and genetic manipulation of RSV infectious clones can be used for analysis of viral replication and pathogenicity mechanisms, as well as for the development of new genetically engineered vaccines. Although previous studies have established reverse genetic manipulation platforms for RSV classic strains long and A2-line19F, no reverse genetic manipulation platforms for recombinant strains that affect pathogenicity through terminal extension of the G gene have been reported to date. Summary of the Invention

[0004] This application constructs two infectious clones with highly homologous genomes but different pathogenicity, providing key biological materials and a genetic manipulation platform for studying the virulence determinants of RSV B subtype. This platform is then used to rescue BA9 genotype variants and their attenuated strains for subsequent studies of in vitro viral replication mechanisms and pathogenicity, neutralizing antibody immune escape, and other research. It also provides a pathogenic technology platform for the development and evaluation of HRSV vaccines, antibodies, and drugs. The details are as follows:

[0005] In the first aspect of the present invention, a reverse genetics system is provided for use in studying RSV virulence-related sequences. The reverse genetics system includes a vector expressing a BA9 genotype variant of RSV and an attenuated strain thereof, wherein the attenuated strain includes a deletion of 7 amino acids at the C-terminus of the G protein of the BA9 genotype variant.

[0006] Preferably, the G protein of the BA9 genotype variant is shown as SEQ ID NO: 3, and the G gene encoding the G protein is shown as SEQ ID NO: 4.

[0007] Preferably, the sequence with 7 amino acids deleted is as shown in SEQ ID NO: 1.

[0008] Preferably, the attenuated strain comprises a deletion of the nucleotide sequence shown in SEQ ID NO: 2 in the G gene of the BA9 genotype variant strain.

[0009] Preferably, the attenuated strain is obtained by amplification using primers.

[0010] Preferably, the primers include the nucleotide sequences shown in SEQ ID NOs: 6 and 7.

[0011] Preferably, the G gene of the attenuated strain is shown as SEQ ID NO: 5.

[0012] Preferably, the sequence deletion reduces the pathogenicity of the BA9 genotype variant.

[0013] Preferably, the application includes obtaining two strains by rescuing vectors expressing the BA9 genotype variant and its attenuated strain respectively.

[0014] Preferably, the two strains have different virulences.

[0015] Preferably, the rescue comprises:

[0016] 1) Transfect the above vector into cells,

[0017] 2) The supernatant obtained by centrifugation after transfection was added to another cell line and cultured;

[0018] 3) Detect virus rescue status.

[0019] Preferably, the vector comprises a nucleotide sequence encoding a BA9 genotype variant or an attenuated strain thereof.

[0020] The vector referred to herein refers to a vector capable of carrying exogenous DNA, mRNA or target gene into host cells for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), Ti plasmid, viral vector (such as retrovirus (including lentivirus), adenovirus, adeno-associated virus, etc.).

[0021] In a specific embodiment, the vector comprises the pBeloBac-11 plasmid.

[0022] Preferably, the vector further comprises regulatory elements, such as a promoter for initiating transcription of the above nucleotide sequence, a terminator for terminating transcription of the above nucleotide sequence, an enhancer sequence or other regulatory sequences that are beneficial to splicing or sequence stability.

[0023] In one embodiment, the regulatory elements include a T7 promoter, a hammerhead ribozyme sequence, and a fragment of a bovine growth hormone (BGH) polyadenylation signal.

[0024] In a specific embodiment, the vector includes a T7 promoter, a hammerhead ribozyme sequence, a gene sequence of RSV BA9 genotype variant or attenuated strain thereof, and a bovine growth hormone polyadenylation signal fragment from the 5' end to the 3' end.

[0025] The cells described herein include host cells (also referred to as recipient cells) and are understood to refer not only to the specific recipient cells, but also to the progeny of such cells. Due to natural, accidental or intentional mutations and / or changes, the progeny may not necessarily be completely identical to the original parent cell, but are still included in the scope of host cells. Suitable cells are known in the art, wherein: the plant cells may be plant cells such as Arabidopsis thaliana, tobacco (Nicotiana tabacum), corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), etc., but are not limited thereto; the animal cells may be mammalian cells (such as Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), baby hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), golden hamster kidney cells (BSR cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, human laryngeal epidermoid carcinoma cells (HEp-2 cells), fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (such as chicken or duck cells), amphibian cells (such as African clawed frog (Xenopus laevis) cells or giant salamander (Andrias davidianus) cells), fish cells (such as grass carp, carp, rainbow trout or catfish cells), insect cells (such as Sf21 cells or Sf-9 cells), etc., but are not limited thereto.

[0026] Preferably, the host cell may also be a microorganism, and the microorganism described herein may be a bacterium, a fungus, an actinomycete, a protozoa, an algae, or a virus. The bacterium may be from the genus Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., etc., but is not limited thereto. For example, the bacterium may be Escherichia coli, Bacillus subtilis, or Bacillus pumilus.

[0027] In a specific embodiment, the cells include TOP10 competent cells, BSRT7-9 cells, and HEp-2 cells.

[0028] In one embodiment, the rescue comprises transfecting the above-mentioned vector into BSRT7-9 cells; after cell transfection, the cells are repeatedly frozen and thawed twice, the entire cell suspension is obtained, centrifuged, the entire supernatant is added to HEp-2 cells, and the cells are continued to be cultured; and the cells are observed for the occurrence of cytopathic effects (CPE) such as syncytia, aggregation, and shedding, and detected using anti-F protein antibodies.

[0029] Preferably, SEQ ID NO: 1 is a virulence-related sequence, and after deleting SEQ ID NO: 1, the virulence of the BA9 mutant virus is weakened.

[0030] In a second aspect, the present invention provides a use of an attenuated RSV BA9 model strain, wherein the G protein of the attenuated RSV BA9 model strain is as shown in SEQ ID NO: 3 with 7 amino acids missing from the C-terminus.

[0031] Preferably, the application includes any one of the following:

[0032] (1) Use in the preparation of products for preventing and / or treating diseases caused by RSV infection;

[0033] (2) Use in the preparation of products for inducing immune responses to RSV antigens;

[0034] (3) Use in preventing and / or treating diseases caused by RSV infection;

[0035] (4) Application in inducing immune response to RSV antigens.

[0036] Preferably, the product comprises a reagent, an antibody, a drug and / or a vaccine. More preferably, the reagent comprises a diagnostic reagent, and the antibody can be used as a diagnostic reagent or a drug.

[0037] The third aspect of the present invention is a method for reducing the pathogenicity of RSV BA9 virus, wherein the method comprises deleting the amino acid shown in SEQ ID NO: 1 at the C-terminus of the G protein of RSV BA9 strain.

[0038] Preferably, the BA9 strain of RSV may be a BA9 genotype variant of RSV, or other BA9 strains.

[0039] In a fourth aspect, the present invention provides a reverse genetics system for RSV strains, comprising a vector expressing a BA9 genotype variant of RSV and an attenuated strain thereof, wherein the attenuated strain comprises a deletion of 7 amino acids at the C-terminus of the G protein of the BA9 genotype variant.

[0040] Preferably, the carrier includes the carrier in the first aspect.

[0041] Preferably, the G protein of the BA9 genotype variant is shown as SEQ ID NO: 3, and the G gene encoding the G protein is shown as SEQ ID NO: 4.

[0042] Preferably, the sequence with 7 amino acids deleted is as shown in SEQ ID NO: 1.

[0043] Preferably, the attenuated strain comprises a deletion of the nucleotide sequence shown in SEQ ID NO: 2 in the G gene of the BA9 genotype variant strain.

[0044] Preferably, the attenuated strain is obtained by amplification using designed primers.

[0045] Preferably, the primers include the nucleotide sequences shown in SEQ ID NOs: 6 and 7.

[0046] Preferably, the G gene of the attenuated strain is shown as SEQ ID NO: 5.

[0047] Preferably, the sequence deletion reduces the pathogenicity of the BA9 genotype variant.

[0048] The fifth aspect of the present invention is a method for preparing the above-mentioned RSV strain reverse genetics system, which comprises inserting a nucleotide sequence encoding a BA9 genotype variant of RSV or an attenuated strain thereof and a regulatory element into a vector.

[0049] Preferably, the nucleotide sequence encoding the attenuated strain deletes a 21 bp nucleotide sequence at the 3' end of the G gene of the RSV BA9 genotype variant.

[0050] Preferably, the method comprises deleting the nucleotide sequence shown in SEQ ID NO: 2.

[0051] Preferably, the method comprises obtaining the strain by amplification through designed primers.

[0052] Preferably, the primers include the nucleotide sequences shown in SEQ ID NOs: 6 and 7.

[0053] The term "include" or "comprising" in the present invention is an open description containing the specified components or steps described, as well as other specified components or steps that do not substantially affect them.

[0054] Beneficial effects of the present invention:

[0055] 1. The two infectious cloned viruses constructed are based on the BA9 genotype variant with highly homologous RSV genomes but different virulence, and its attenuated strain Δ7aa. The attenuated strain Δ7aa has 7 amino acids deleted, resulting in reduced virulence.

[0056] 2. The method for constructing infectious clones is simple and convenient, allowing direct DNA transfection without the need for in vitro transcription into viral RNA for transfection. This overcomes the shortcomings of RNA in vitro, such as instability and susceptibility to degradation. Furthermore, the self-cleavage function of the hepatitis D virus ribozyme is utilized to precisely target the 3' end of the RSV genome for infectious clones, providing a fast, simple, and precise platform for RSV genetic manipulation.

[0057] 3. The RSV infectious clone platform obtained can be applied to the study of in vitro viral replication and pathogenicity mechanisms, neutralizing antibody immune escape, and can also be used for the subsequent development of safer and more efficient new RSV genetically engineered live vaccines; it provides a pathogenic technology platform for antibodies and drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Schematic diagram of vector construction.

[0059] Figure 2 The following are the cytopathic effects: A is the BA9 genotype variant strain lesion, B is the attenuated strain Δ7aa lesion, C is the laboratory reference strain 9320 lesion, and D is the normal cell control group.

[0060] Figure 3 Figure 3 is the immunofluorescence result, where A is the immunofluorescence of BA9 genotype variant, B is the immunofluorescence of attenuated strain Δ7aa, C is the immunofluorescence of laboratory reference strain 9320, and D is the normal cell control group.

[0061] Figure 4 These are the neutralization antibody inhibition curves, where A is the neutralization curve of the BA9 genotype variant, and B is the neutralization curve of the attenuated strain Δ7aa.

[0062] Figure 5 The weight changes of mice after infection.

[0063] Figure 6 This is the viral load in the lungs of mice after infection. DETAILED DESCRIPTION

[0064] To help those skilled in the art better understand the technical solutions of the present invention, the following clearly and completely describes the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0065] Unless otherwise specified, the materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources.

[0066] The conventional experimental methods in the following examples are described in Sambrook et al., Molecular Cloning Experimental Manual, 3rd edition (Beijing: Science Press, 2002). The use of the instruments is described in the instrument operating instructions.

[0067] In the embodiment of the present invention, the virus includes BA9 genotype variant strain WT and attenuated strain Δ7aa, and the cells include BSRT7-9 and HEp-2 cell lines.

[0068] In the embodiments of the present invention, plasmids and strains: pBeloBac-11 plasmid was stored in this laboratory, and Top10 competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0069] In the examples of the present invention, other reagents used: RNase Free H2O, Mut Express II Fast Mutagenesis Kit V2 were purchased from Novagen; plasmid extraction kit was purchased from QIAGEN Co., Ltd.; DMEM medium was purchased from Hyclone Biochemical Products Co., Ltd.; fetal bovine serum was purchased from Gibco; DyLight 488 was purchased from Invitrogen; and Lipofectamine™ 3000 Transfection Reagent was purchased from Invitrogen.

[0070] Example 1 Construction and rescue of full-length cDNA clones of the BA9 genotype variant strain WT and attenuated strain Δ7aa

[0071] 1.1. Construction of full-length BA9 genotype variant (WT) vector

[0072] As shown in the vector construction schematic in Figure 1, the replica plasmid pBeloBac-11 was selected as the primary vector. A fragment containing the T7 promoter, Hammerhead ribozymes (HHR), the full-length WT gene, and the polyadenylation signal (polyA) of bovine growth hormone (BGH) was inserted into the pBeloBac-11 vector after the BamHI restriction enzyme site. The recombinant plasmid was synthesized by Nanjing GenScript and verified by sequencing, named pBeloBac-WT.

[0073] 1.2Δ7AA primer design

[0074] Based on the WT full gene sequence (SEQ ID NO: 8), a pair of point mutation amplification primers were designed using CE Design (as shown in Table 1) to delete the extended 7 amino acids (SEQ ID NO: 1). The corresponding nucleotide sequence is SEQ ID NO: 2, the WT G protein amino acid sequence is shown in SEQ ID NO: 3, the WT G gene nucleotide sequence is shown in SEQ ID NO: 4, and the Δ7AA G gene nucleotide sequence is shown in SEQ ID NO: 5.

[0075] Table 1. Primers used to amplify the full RSV gene in constructing infectious clones of the present invention

[0076]

[0077] 1.3 Construction of infectious clone of attenuated strain (Δ7aa)

[0078] 1.3.1 Full-gene amplification of the Δ7aa infectious clone

[0079] Refer to the instructions of Mut Express II Fast Mutagenesis Kit V2, use WT as template, and amplify the full gene sequence with Δ7aa-F and Δ7aa-R pairs. The specific steps are as follows:

[0080] The PCR reaction system was as follows: 1 ng DNA template, 2 μL each of upstream and downstream primer pairs (10 μM), 1 μL of Phanta Max Super-Fidelity DNA Polymerase, 25 μL of 2×Max Buffer, 1 μL of dNTP Mix (10 mM each), and RNase-Free H2O to 50 μL.

[0081] The PCR reaction program was: 95°C for 30 s, 95°C for 15 s, 65°C for 15 s, and 72°C for 12 min, for 30 cycles.

[0082] Amplify the full Δ7aa gene as described above. Then, add 1 μL of Dpn I, gently pipette to mix, and briefly centrifuge to collect the residue at the bottom of the tube. Incubate at 37°C for 1-2 hours. The digestion product can be used directly in the recombination reaction without purification.

[0083] Reconstitution reaction: Prepare the following reaction system on ice:

[0084] Table 2 Reaction system

[0085] Components Recombination reaction Negative control a Dpn I digestion products 400ng 400ng 5×CE II Buffer 4 μl 0μl Exnase II 2 μl 0μl <![CDATA[ddH2O]]> Up to 20μl Up to 20μl

[0086] Use a pipette to gently pipette to mix, centrifuge briefly to collect the reaction solution at the bottom of the tube, and incubate at 37°C for 30 minutes before immediately cooling on ice.

[0087] Take 2 μl of the ligation product and transform it into TOP10 competent cells. Pick independent colonies for pure culture and perform PCR testing using detection primers. Select PCR-positive bacteria and culture them overnight. Extract plasmid DNA and verify the full-length sequence of the plasmid using next-generation sequencing.

[0088] 1.4 Rescue of WT and Δ7AA infectious clone viruses

[0089] BSRT7-9 cells were cultured in DMEM medium containing 10% FBS at a volume of 2 × 10 6 The cells were seeded into 6-well cell culture plates at a density of cells / well and cultured in a 37°C, 5% CO2 incubator until the cell density reached about 80%. The culture medium was then replaced with DMEM containing 2% FBS. TM 3000 Transfection Reagent instructions for cell transfection, the specific steps are as follows:

[0090] First, prepare the plasmid premix in one EP tube: 500 ng of infectious clone plasmid, 1 μL of P3000, and 25 μL of OPTI MEM; prepare the Lipo3000 premix in another EP tube: 2 μL of Lipo3000 and 25 μL of OPTI MEM. Finally, mix the premixes obtained in the above two steps, let them stand at room temperature for 15 minutes, and add them to the cells to be transfected.

[0091] 120 hours after cell transfection, freeze the entire culture plate at -80°C, freeze and thaw twice, take out the entire cell suspension, centrifuge at 10,000×g for 1 minute. Take all the supernatant and add it to HEp-2 cells, continue to culture, and observe the cell condition every day. Figure 2 As shown in the figure, 3 days after the rescue virus, cells showed syncytia, aggregation, shedding and other cytopathic effects (CPE), while negative control cells showed no obvious CPE. The CPE characteristics of WT and Δ7AA rescued viruses were basically consistent with those of the laboratory strain (9320, GenBank: AY353550). Figure 3 As shown, the fifth generation rescued virus was tested with RSV anti-F protein palivizumab, and specific green fluorescence was observed in the laboratory strain (9320) and the rescued virus, while no fluorescence was produced in the negative control group, which again verified that the virus rescue was successful.

[0092] Virus neutralization test measures the neutralizing ability of different strains to neutralizing antibodies:

[0093] WT and Δ7AA were incubated with serially diluted Nissir monoclonal antibody at 37°C for 1 hour. The viral titer after incubation was determined using HEp-2 cells. The specific operation was to extract viral RNA 48 hours after infection and reverse transcribe and determine the viral copy number by fluorescence quantitative PCR. The dose response curve was measured by antibody neutralization assay. Figure 4 As shown, the neutralization EC50 of WT and Nicevir monoclonal antibody is 2.80 ng / mL, and the neutralization EC50 of Δ7AA and Nicevir monoclonal antibody is 0.83 ng / mL. The results show that compared with WT, the toxicity of Δ7AA is greatly reduced, and there is a significant difference between the two.

[0094] Example 2 Mouse challenge experiment

[0095] The specific experimental methods are as follows:

[0096] 1. Weighing:

[0097] BALB / c mice aged 6 to 8 weeks were randomly divided into four groups, including the laboratory reference strain 9320 group, the BA9 genotype variant (WT) group, the attenuated strain (Δ7AA) group, and a negative control group (MOCK), with 6 mice in each group. During the experiment, mice were anesthetized with isoflurane for challenge and sampling. Each mouse in each challenge group was intranasally inoculated with 5.0×10 5 PFU / 50μL. In the negative control group, mice were intranasally dripped with the same volume of DMEM as a control. The mice were weighed daily and killed on the 3rd, 4th and 5th days to collect their lung tissues.

[0098] 2. Determination of lung virus titer

[0099] Place grinding steel beads in a grinding tube, add 0.1 g of RSV-infected mouse lung tissue and 0.1 mL of PBS buffer, place it in an automated crusher, grind at 25 Hz for 1 min, centrifuge at 5000 rpm for 10 min, take the supernatant, extract the nucleic acid, and use fluorescent quantitative PCR to determine the viral load of HRSV in the mouse lungs.

[0100] The experimental results are as follows Figure 5 and 6 As shown, the weight changes of mice in the WT and Δ7AA groups after infection were similar, reaching the lowest value on the third day, and the WT group had a lower body weight. The lung viral load in the WT group was significantly different from that in the 9320 group and the Δ7AA group on the third day, indicating that the virulence of the Δ7AA group was weaker than that of the WT group.

[0101] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and substance of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be readily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. Application of a reverse genetics system in studying virulence-related sequences of respiratory syncytial virus (RSV), characterized in that: The reverse genetic system includes a vector expressing the BA9 genotype variant of RSV or its attenuated strain, wherein the attenuated strain is a vector that lacks 7 amino acids from the last amino acid at the C-terminus of the G protein of the BA9 genotype variant. The nucleotide sequence of the BA9 genotype variant is shown in SEQ ID NO: 8, and the G protein of the BA9 genotype variant is shown in SEQ ID NO:

3.

2. The use according to claim 1, characterized in that The sequence lacking 7 amino acids is shown in SEQ ID NO:

1.

3. The use according to claim 2, characterized in that The sequence deletion reduces the pathogenicity of the BA9 genotype variant strain.

4. The use according to claim 1, characterized in that The application includes obtaining two strains by rescuing the vector expressing the BA9 genotype variant or the attenuated strain thereof.

5. The use according to claim 4, characterized in that Said rescue includes: 1) transfecting the vector into cells, 2) Add the supernatant obtained by centrifugation after transfection to another cell line for further culture; 3) Detect virus rescue status.

6. The use according to claim 5, characterized in that The vector comprises a nucleotide sequence encoding a BA9 genotype variant of RSV or an attenuated strain thereof and a regulatory element.

7. A method for reducing the pathogenicity of a BA9 genotype variant of respiratory syncytial virus (RSV), characterized in that: The method is to delete the amino acid shown in SEQ ID NO: 1 at the C-terminus of the G protein of the BA9 genotype variant of RSV, and the nucleotide sequence of the BA9 genotype variant is shown in SEQ ID NO:

8.

8. A reverse genetics system for respiratory syncytial virus (RSV), characterized in that: The reverse genetic system includes a vector expressing the BA9 genotype variant of RSV or its attenuated strain, wherein the attenuated strain is a vector that lacks 7 amino acids from the last amino acid at the C-terminus of the G protein of the BA9 genotype variant. The nucleotide sequence of the BA9 genotype variant is shown in SEQ ID NO: 8, and the G protein of the BA9 genotype variant is shown in SEQ ID NO:

3.

9. The method for preparing the reverse genetics system according to claim 8, characterized in that: The method comprises inserting a nucleotide sequence encoding a BA9 genotype variant of RSV or an attenuated strain thereof into a vector.

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