Establishment of multiplex detection method for influenza virus neutralizing antibodies based on fluorescent pseudovirus

Abstract

The present application relates to the technical field of biotechnology, in particular to a vector, a vector combination and a pseudovirus prepared using the same. The present application also relates to a method for detecting the neutralization activity of an influenza virus antibody and a method for screening a candidate drug capable of inhibiting the infection of cells by an influenza virus.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to a vector, a combination of vectors, and pseudoviruses prepared using the same. This application also relates to a method for detecting the neutralizing activity of influenza virus antibodies, and a method for screening candidate drugs capable of inhibiting influenza virus infection of cells. Background Technology

[0002] Influenza, commonly known as the flu, is a highly contagious acute respiratory illness caused by the influenza virus. After infection, people typically experience asymptomatic or mild symptoms such as cough, sore throat, runny nose, fever, headache, and muscle aches. Severe cases can lead to viral pneumonia and even acute respiratory failure. According to the WHO, influenza epidemics cause approximately 4 million severe cases and 500,000 deaths annually, making it the second leading cause of death after acquired immunodeficiency syndrome (AIDS).

[0003] Influenza vaccination is one of the most effective and cost-efficient strategies for preventing and controlling influenza outbreaks. Currently, influenza vaccines approved for human use in various countries fall into three main categories: inactivated vaccines, live attenuated vaccines, and recombinant HA influenza vaccines. Regardless of the type, they all contain components of three circulating strains of influenza A virus (H1N1, H3N2) and influenza B virus (Victoria or Yamagata lineage). In recent years, to improve the efficacy of seasonal influenza vaccines, quadrivalent influenza vaccines have been approved by the FDA, containing both Victoria and Yamagata lineages of influenza B virus.

[0004] Currently, hemagglutination inhibition assays, micro-neutralization assays, and pseudovirus neutralization assays are commonly used to detect serum neutralizing antibody levels after influenza vaccination, with antibody titers used to evaluate immunization efficacy. However, hemagglutination inhibition assays are prone to false positives, micro-neutralization assays require live virus handling, and pseudovirus neutralization assays typically use luciferase labeling, making the detection process cumbersome and the substrates expensive. Furthermore, these three methods share a common limitation: each plate can only detect one type of neutralizing antibody, making high-throughput detection of multiple antibodies impossible. Therefore, establishing a safe, convenient, and high-throughput detection method for simultaneously detecting multiple influenza virus neutralizing antibodies in serum is of great significance. This study utilized readily detectable, highly sensitive, and broadly categorized fluorescent proteins to construct various fluorescent backbone plasmids, which were then packaged with influenza virus HA membrane plasmids to form influenza vaccine strain pseudoviruses of different colors, establishing a multi-type detection method for influenza virus neutralizing antibodies based on fluorescent pseudoviruses. This method can simultaneously detect multiple neutralizing antibodies present in serum after influenza vaccination, offering safety, reliability, convenience, and high-throughput detection. Summary of the Invention

[0005] The applicant of this application has conducted extensive experiments and provided a fluorescent backbone plasmid for preparing pseudoviruses. The plasmid of this application can be efficiently packaged with HA (e.g., H1, H3, BV, and BY subtypes) membrane protein plasmids to obtain pseudoviruses. The obtained pseudoviruses have strong infectivity, produce stable fluorescence, and can accurately detect neutralizing antibodies. Furthermore, this application also provides pseudovirus combinations that can simultaneously detect multiple subtypes of neutralizing antibodies present in serum after influenza vaccination, achieving high-throughput detection.

[0006] Therefore, in a first aspect, this application provides a vector that, based on the pSG3Δenv vector, contains an exogenous sequence as shown in SEQ ID NO: 29, or has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 29, or has a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to SEQ ID NO: 29.

[0007] In some embodiments, the pSG3Δenv vector comprises or has a nucleotide sequence as shown in SEQ ID NO: 23, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with it.

[0008] In some embodiments, the exogenous sequence is inserted in the pSG3Δenv vector at the position between bases 13526 and 13527 of the nucleotide sequence corresponding to SEQ ID NO:23.

[0009] As used herein, “pSG3Δenv” is an HIV-1 virus-based backbone plasmid vector. The pSG3Δenv plasmid is genetically engineered to disrupt the env gene, preventing the expression of the viral envelope protein (Env). pSG3Δenv can be used to generate pseudoinfectious viruses and facilitates immunological and pharmacological analyses. In some embodiments, the nucleotide sequence of the pSG3Δenv vector is shown in SEQ ID NO: 23.

[0010] The vector described in this application can be used as a standalone vector product to prepare pseudoviruses expressing fluorescent proteins. The prepared fluorescent pseudoviruses can be used for neutralization assays to rapidly assess the neutralizing effect of serum from naturally infected and vaccinated individuals on the virus. Furthermore, the vector of this application exhibits excellent assembly capabilities with various subtypes of membrane proteins (e.g., the HA protein of influenza virus), resulting in pseudoviruses with high titers and high detection sensitivity. By combining it with different HA proteins, different subtypes of pseudoviruses (e.g., pseudoviruses of influenza virus) can be conveniently prepared.

[0011] In a second aspect, this application provides a vector combination comprising the vector described above, and an influenza virus HA (Hemagglutinin) protein expression plasmid.

[0012] In some embodiments, the influenza virus HA protein expression plasmid contains a nucleotide sequence encoding an H1 protein, H3 protein, BV protein, or BY protein.

[0013] In some embodiments, the influenza virus HA protein expression plasmid contains a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27.

[0014] This application verifies the ability of the vector of this application to assemble with the HA protein of multiple subtypes of influenza virus, wherein the vector of this application exhibits significantly higher titers and detection sensitivity when packaged with BV protein-containing pseudoviruses. Therefore, in some embodiments, the influenza virus HA protein expression plasmid comprises a nucleotide sequence encoding the amino acid sequence of the BV protein. In some embodiments, the influenza virus HA protein expression plasmid comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the influenza virus HA protein expression plasmid comprises a sequence encoding at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO: 26, or a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to SEQ ID NO: 26. The term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap may be introduced in the first amino acid sequence or nucleic acid sequence to best align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions / total number of positions × 100%).

[0015] In a third aspect, this application provides a host cell comprising the vector or a combination of vectors as described above.

[0016] In some embodiments, the host cell is a human cell, such as a hematopoietic cell, epithelial cell, hepatocyte, tumor cell, or nerve cell.

[0017] In some implementations, the host cell is a HEK 293T cell.

[0018] In a fourth aspect, this application provides a method for preparing a pseudovirus, comprising:

[0019] (1) Transfecting the vector or combination of vectors as described above into the host cells as described above; and

[0020] (2) Harvesting fake viruses.

[0021] In some implementations, the method is performed by the following steps:

[0022] (a) Provide the vector as described above, and the influenza virus HA protein expression plasmid;

[0023] (b) Transfect the vector and influenza virus HA protein expression plasmid as described above into the host cells as described above; and

[0024] (c) Harvesting fake viruses.

[0025] In some embodiments, the mass ratio of the vector described above to the influenza virus HA protein expression plasmid is 4:1 to 1:1 (e.g., 3:1, 2:1).

[0026] In some embodiments, the method further includes treating the harvested pseudovirus with TPCK trypsin (e.g., 40-60 µg / ml or 60-80 µg / ml of TPCK trypsin).

[0027] In some embodiments, the influenza virus HA protein expression plasmid comprises a nucleotide sequence encoding the amino acid sequence of the H3 protein. In some embodiments, the influenza virus HA protein expression plasmid comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:25.

[0028] In a fifth aspect, this application provides a pseudovirus prepared by the method described above.

[0029] In some embodiments, the pseudovirus comprises a protein having amino acid sequences as shown in SEQ ID NO: 32 and SEQ ID NO: 26.

[0030] In some embodiments, the pseudovirus comprises a protein having an amino acid sequence as shown in SEQ ID NO: 32 and SEQ ID NO: 24.

[0031] In some embodiments, the pseudovirus comprises a protein having an amino acid sequence as shown in SEQ ID NO: 32 and SEQ ID NO: 25.

[0032] In some embodiments, the pseudovirus comprises a protein having the amino acid sequences shown in SEQ ID NO: 32 and SEQ ID NO: 27.

[0033] This application, through extensive experiments, investigated the compatibility of different fluorescent proteins and compared the packaging efficiency of different fluorescent proteins with different subtypes of HA protein, as well as the titers and detection sensitivity of the resulting pseudoviruses. It discovered that other pseudoviruses expressing specific fluorescent proteins can be used in combination with the pseudoviruses prepared in this application. These pseudoviruses each have different subtypes and contain different fluorescent proteins, allowing them to be mixed in the same system while their individual fluorescence signals can be distinguished. For example, when using influenza virus pseudoviruses to detect neutralizing antibodies, these pseudoviruses can be used in conjunction to detect serum antibodies, simultaneously detecting antibodies of different subtypes in the serum.

[0034] Therefore, in a sixth aspect, this application provides a combination of fake viruses, which includes the fake virus as described above, as well as other fake viruses.

[0035] In some embodiments, the pseudovirus combination comprises: a first pseudovirus containing a protein having the amino acid sequences shown in SEQ ID NO: 32 and SEQ ID NO: 26; and a second pseudovirus containing a protein having the amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 33.

[0036] In some embodiments, the pseudovirus combination further includes a third pseudovirus comprising a protein having the amino acid sequences shown in SEQ ID NO: 25 and SEQ ID NO: 31.

[0037] In a seventh aspect, this application provides a method for detecting the neutralizing activity of influenza virus antibodies in a sample, the method comprising contacting the sample with the pseudovirus or pseudovirus combination before, simultaneously with, or after contacting the pseudovirus or pseudovirus combination as described above with a host cell.

[0038] In some implementations, the method includes:

[0039] (1) Contact the sample with the pseudovirus as described above or a combination of pseudoviruses as described above;

[0040] (2) Introduce the pseudovirus or pseudovirus combination obtained in (1) into the host cell;

[0041] (3) Under conditions where fluorescence is emitted, observe the host cells, count the number of positive cells in the host cells, and calculate the ID. 50 The value was used to detect the neutralizing activity of the influenza virus antibodies.

[0042] In some implementations, the method is performed by the following steps:

[0043] (a) The first fake virus, the second fake virus and the third fake virus are brought into contact with the same sample (e.g., the first fake virus, the second fake virus and the third fake virus are brought into contact with the same sample simultaneously).

[0044] (b) Take the sample from step (a) and bring it into contact with the host cells;

[0045] (c) Under conditions where the fluorescence of the first pseudovirus, the second pseudovirus, and the third pseudovirus can be detected, the host cells are observed, the number of positive cells in the host cells is counted, and the ID is calculated. 50 The value was used to detect the neutralizing activity of the influenza virus antibodies.

[0046] In some implementations, in step (a), the antibody binds to the first pseudovirus, the second pseudovirus, and / or the third pseudovirus, rendering them incapable of infecting the host cell.

[0047] In some implementations, in step (a), the amounts of the first fake virus, the second fake virus, and the third fake virus are each independently selected from 1TCID. 50 -5TCID 50 .

[0048] In some embodiments, in step (b), the sample from step (a) is contacted with host cells for 24–48 h or 48–144 h (e.g., 48 h, 72 h, 86 h, 120 h, 144 h).

[0049] In some embodiments, in step (b), the amount of host cells is 30,000 to 60,000 per well (e.g., 40,000 or 50,000 per well).

[0050] In some embodiments, in step (c), the fluorescence of the first pseudovirus is detected using a Texas Red excitation source. In some embodiments, in step (c), the fluorescence of the second pseudovirus is detected using a CFP excitation source. In some embodiments, in step (c), the fluorescence of the third pseudovirus is detected using a YFP or RFP excitation source.

[0051] In some embodiments, the sample contains influenza virus antibodies against multiple subtypes of influenza virus (e.g., anti-H1 subtype antibodies, anti-H3 subtype antibodies, and / or anti-BV subtype antibodies). Furthermore, due to the declining prevalence of BY influenza virus in recent years, trivalent vaccines targeting H1, H3, and BV are expected to become the mainstream in the future. Therefore, the combined detection of the first, second, and third pseudoviruses can simultaneously detect the neutralizing activity of influenza virus antibodies in the sample.

[0052] In some embodiments, the host cell is a mammalian cell, such as a human cell, a dog cell, or a monkey cell.

[0053] In some implementations, the host cell is an MDCK cell.

[0054] In some embodiments, the sample is selected from plasma, serum, or any combination thereof.

[0055] In an eighth aspect, this application provides a method for screening candidate drugs capable of inhibiting influenza virus infection of cells, the method comprising contacting the candidate drug with the pseudovirus or pseudovirus combination before, simultaneously with, or after contacting the pseudovirus or pseudovirus combination as described above with the host cell.

[0056] In some implementations, the method includes:

[0057] (1) Contact the candidate drug with the pseudovirus as described above or a combination of pseudoviruses as described above;

[0058] (2) Introduce the pseudovirus or pseudovirus combination obtained in (1) into the host cell;

[0059] (3) Under conditions where fluorescence is emitted, observe the host cells to determine whether the candidate drug can inhibit influenza virus infection of cells.

[0060] In some implementations, the method is performed by the following steps:

[0061] (a) Contact the first pseudovirus, the second pseudovirus, and the third pseudovirus with the candidate drug;

[0062] (b) Take the sample from step (a) and bring it into contact with the host cells;

[0063] (c) Under conditions where the fluorescence of the first pseudovirus, the second pseudovirus, and the third pseudovirus can be detected, the host cells are observed, the number of positive cells in the host cells is counted, and the ID is calculated. 50 The value is used to evaluate the drug's ability to inhibit influenza virus infection of cells. In some embodiments, the host cell is a mammalian cell, such as human cells, dog cells, or monkey cells.

[0064] In some implementations, the host cell is an MDCK cell.

[0065] In some embodiments, in step (a), the drug renders the first pseudovirus, the second pseudovirus, and / or the third pseudovirus incapable of infecting the host cell.

[0066] In some implementations, in step (a), the amounts of the first fake virus, the second fake virus, and the third fake virus are each independently selected from 1TCID. 50 -5TCID 50 .

[0067] In some embodiments, in step (b), the drug from step (a) is exposed to host cells for 1–24 h, 24–48 h, or 48–144 h (e.g., 48 h, 72 h, 86 h, 120 h, 144 h).

[0068] In some embodiments, in step (b), the amount of host cells is 30,000 to 60,000 per well (e.g., 40,000 or 50,000 per well).

[0069] In some embodiments, in step (c), the fluorescence of the first pseudovirus is detected using a Texas Red excitation source. In some embodiments, in step (c), the fluorescence of the second pseudovirus is detected using a CFP excitation source. In some embodiments, in step (c), the fluorescence of the third pseudovirus is detected using a YFP or RFP excitation source.

[0070] Terminology Definition

[0071] As used herein, the term "HA protein" refers to hemagglutinin (HA), a structural protein in influenza viruses. The genomes of influenza A and B viruses primarily encode eight structural proteins and two non-structural proteins. The structural proteins include polymerase protein (PB), hemagglutinin (HA), neuraminidase (NA), matrix protein (M), and nucleoprotein (NP), while the non-structural proteins include NS1 and NS2. HA protein is an I-type transmembrane glycoprotein, existing as a homotrimer. HA can bind to sialic acid receptors on the host cell surface and mediate virus-host membrane fusion, allowing the virus to adsorb onto the cell surface and enter the cell; it is also a major antigen on the viral surface and a primary target of the host immune response.

[0072] Based on differences in nucleoproteins and matrix proteins, influenza viruses can be classified into four types: influenza A virus, influenza B virus, influenza C virus, and influenza D virus. Influenza A virus, based on differences in its surface glycoproteins HA and NA, can be further divided into 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11). Among these, subtypes H1-H16 and N1-N9 were isolated from waterfowl and belong to the traditional influenza A virus category; their HA and NA proteins on the viral membrane surface can recognize sialic acid receptors. According to genetic evolution, the 18 HA subtypes of influenza A can be further divided into two groups: Group 1 includes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18; Group 2 includes H3, H4, H7, H10, H14, and H15. The HA protein of influenza B virus does not have subtypes, but rather exists in two different lineages: B / Victoria / 2 / 1987-like (BV) and B / Yamagata / 16 / 1988-like (BY). In some embodiments, the HA protein is an H1, H3, BV, or BY protein.

[0073] As used herein, the terms "pseudovirus" and "virus-like virus" have the same meaning and are used interchangeably; they refer to a virus-like particle formed by the self-assembly of viral proteins, which either does not encapsulate nucleic acid or encapsulates other nucleic acids. Therefore, while the pseudovirus or virus-like virus can infect host cells, it does not have the ability to replicate autonomously. Consequently, it has higher biosafety compared to a real virus. The packaging system of a pseudovirus generally consists of two parts: a packaging component and an expression component. The packaging component is constructed from the viral genome (e.g., HIV-1) with the genetic information required for packaging, reverse transcription, and integration removed, providing the proteins necessary for the pseudovirus particle. The expression component is complementary to the packaging component, containing the genetic information required for packaging, reverse transcription, and integration, as well as the exogenous target gene. By co-transfecting host cells with the packaging component and the vector component, pseudovirus particles can be harvested from the cell supernatant.

[0074] In some embodiments, the packaging component comprises or consists of a backbone plasmid. In some embodiments, the expression component comprises or consists of an influenza virus membrane protein expression plasmid. In some embodiments, the influenza virus membrane protein expression plasmid is also called an HA membrane protein plasmid, i.e., an expression plasmid expressing the influenza virus HA protein.

[0075] As used in this article, the term "exogenous" or "heterogeneous" refers to a nucleotide or amino acid sequence that is not found in the native nucleic acids or proteins of a given organism.

[0076] As used herein, the terms "backbone plasmid" and "packaging plasmid" have the same meaning and are used interchangeably. As is generally understood by those skilled in the art, a viral vector system (particularly a lentiviral vector system) can consist of two parts: a packaging component (e.g., a backbone plasmid) and a vector component (e.g., a recombinant expression vector carrying a target gene); wherein the packaging component (e.g., the backbone plasmid) provides all the accessory proteins required for transcription and packaging of genetic material into recombinant pseudoviral particles. Thus, high-titer pseudoviral particles can be generated by co-transfecting cells with a recombinant expression vector and a backbone plasmid, followed by pseudoviral packaging within the cells, and subsequently secretion of the packaged pseudoviral particles into an extracellular culture medium. Such packaging plasmids or backbone plasmids are well known to those skilled in the art, for example, the HIV-1-based backbone plasmid pSG3Δenv. In some embodiments, the backbone plasmid is the fluorescent backbone plasmid of this application. In some embodiments, the recombinant expression vector is the influenza virus membrane protein expression plasmid HA membrane protein plasmid.

[0077] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0078] As used herein, the term “host cell” refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK21 cells, HEK 293T cells, or human cells.

[0079] Those skilled in the art will understand that the design of expression vectors can depend on factors such as the choice of host cells to be transformed and the desired expression level. A vector can be introduced into a host cell to produce transcripts, proteins, or peptides, including proteins, fusion proteins, isolated nucleic acid molecules, etc., as described herein.

[0080] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap may be introduced in the first amino acid sequence or nucleic acid sequence to best align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. The percentage identity between two sequences is a function of the number of identity positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions / total number of positions × 100%). In some embodiments, the two sequences are of the same length.

[0081] As used herein, the term "corresponding position" refers to the amino acid position at the equivalent position in the two sequences being compared when performing an optimal alignment, i.e., when the two sequences are aligned to obtain the highest percentage identity. For example, the expression "corresponding to the position between bases 13526 and 13527 of the nucleotide sequence shown in SEQ ID NO: 23" means the position between bases in the compared sequence that are at the equivalent position to bases 13526 and 13527 of SEQ ID NO: 23 when performing an optimal alignment, i.e., when the sequence is aligned with SEQ ID NO: 23 to obtain the highest percentage identity.

[0082] As used herein, the term "effective amount" means an amount that is effective in achieving the intended purpose. For example, an effective amount for the prevention or treatment of a disease (e.g., rotavirus infection) means an amount that is effective in preventing, stopping, or delaying the onset of a disease (e.g., rotavirus infection), or in alleviating, reducing, or treating the severity of an existing disease (e.g., a disease caused by rotavirus infection). Determining such an effective amount is within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0083] As used herein, the term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment to bind to antigenic proteins on a virus, thereby preventing the virus from infecting cells and / or maturing and / or releasing viral progeny. Antibodies or antibody fragments with neutralizing activity can prevent viral amplification, thereby inhibiting or eliminating viral infection.

[0084] As used in this article, the term "MOI (multiplicity of infection)" refers to the ratio of virus to cell count when a virus infects a host cell. MOI is generally considered a ratio without units, although its implicit unit can be pfu number / cell. Choosing an appropriate MOI value can improve viral infection efficiency.

[0085] Beneficial effects of the invention

[0086] The applicant of this application has conducted extensive experiments and provided a fluorescent backbone plasmid for preparing pseudoviruses. The plasmid of this application can efficiently package with HA (e.g., H1, H3, BV, and BY subtypes) membrane protein plasmids to obtain pseudoviruses. The obtained pseudoviruses have strong infectivity, produce stable fluorescence, and can accurately detect neutralizing antibodies. Among these, the plasmid of this application shows the highest assembly efficiency with BV subtype HA membrane proteins, resulting in the highest titer of the obtained pseudoviruses.

[0087] Furthermore, this application also provides pseudovirus combinations that can simultaneously detect multiple subtypes of neutralizing antibodies present in serum after influenza vaccination, achieving high-throughput detection with advantages such as high detection sensitivity, ease of operation, and savings in manpower and resources. For example, the combination of H1-AmCyan, H3-Cherry, and BV-Yellow pseudoviruses provided in this application can simultaneously detect neutralizing antibodies against three subtypes of influenza virus—H1, H3, and BV—in a sample, significantly improving detection efficiency. Simultaneously, this method exhibits extremely high correlation with the results of the traditional Fluc neutralization assay, and the detected antibody titers are at least twice as high (e.g., at least twice as high) as those detected by the traditional Fluc neutralization assay.

[0088] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0089] Figure 1 The results show the amplification of different fluorescent fragments. Among them, Figure 1 A shows the amplification results of CMV-AmCyan, CMV-ZsYellow, CMV-ZsGreen, mOrange, E2 Crimson, pdTomato, and mCherry; Figure 1 B shows the CMV amplification results ligated with mOrange, E2Crimson, pdTomato, and mCherry, respectively; Figure 1 C shows the amplification results of CMV-mOrange, CMV-E2 Crimson, CMV-pdTomato, and CMV-mCherry.

[0090] Figure 2 The structure of the fluorescent backbone plasmid SG3-AmCyan is shown as an example. Similarly, the structures of the fluorescent backbone plasmids SG3-ZsGreen, SG3-ZsYellow, SG3-mOrange, SG3-E2 Crimson, SG3-pd Tomato, and SG3-mCherry are the same as those of SG3-AmCyan, except that the sequences indicated by the arrows (blue) are replaced with the corresponding promoters and nucleotide sequences encoding fluorescent proteins.

[0091] Figure 3 This displays the results of the selection of enzyme digestion reagents for pseudoviruses. The horizontal axis represents the different enzyme digestion methods for each subtype, and the vertical axis represents the number of fluorescent spots at a 3-fold dilution of the pseudovirus when observing the titration results.

[0092] Figure 4 The results of selecting enzyme digestion reagent concentrations are displayed. The horizontal axis represents different TPCK trypsin concentrations, and the vertical axis represents the number of fluorescent spots at a 3-fold dilution of the pseudovirus when observing the titration results.

[0093] Figure 5 This displays the results of the selected transfection ratio.

[0094] Figure 6 This diagram illustrates the identification of sensitive cells in the in vitro neutralizing antibody detection method. The horizontal axis represents different infected cells, and the vertical axis represents the number of fluorescent spots at a 3-fold dilution of the pseudovirus when observing the titration results.

[0095] Figure 7 This diagram illustrates the determination of MDCK cell count in the in vitro neutralizing antibody detection method. The horizontal axis represents different MDCK cell counts, and the vertical axis represents the number of fluorescent spots at a 3-fold dilution of the pseudovirus when observing the titration results.

[0096] Figure 8 This illustrates the determination of detection time in the in vitro neutralizing antibody detection method. The horizontal axis represents different detection times, and the vertical axis represents the number of fluorescent spots at a 3-fold dilution of the pseudovirus when observing the titration results.

[0097] Figure 9 This demonstrates the determination of the amount of pseudovirus added in the in vitro neutralizing antibody detection method.

[0098] Figure 10 This displays the validation results of the sensitivity of the in vitro neutralizing antibody detection method. The horizontal axis represents the logarithm of the pseudovirus dilution factor, and the vertical axis represents the serum inhibition rate.

[0099] Figure 11 This displays the validation results of the specificity of the in vitro neutralizing antibody detection method. The horizontal axis represents different pseudovirus subtypes, and the vertical axis represents the ID. 50 .

[0100] Figure 12 This displays the results of detecting single-subtype pseudoviruses with single-subtype and mixed-subtype antisera. The horizontal axis represents different antisera, and the vertical axis represents lgID. 50 .

[0101] Figure 13 This displays the results of detecting single-subtype pseudoviruses and mixed-subtype pseudoviruses in post-clinical immunization serum. The horizontal axis represents pseudoviruses, and the vertical axis represents lgID. 50 .

[0102] Figure 14 This displays the results of detecting single-subtype and three-subtype pseudoviruses in post-clinical immunization serum. The horizontal axis represents pseudoviruses, and the vertical axis represents lgID. 50 .

[0103] Figure 15 This displays the results of a repeatability test of clinically immunized serum after mixing three subtypes of pseudoviruses. The experiment used six immunized sera, with each row representing the result of one immunized serum sample. The horizontal axis, assays 1-3, represents three replicates performed at three different time points. Each assay has four values ​​representing four replicates per repetition.

[0104] Figure 16 This paper presents a comparison between the fluorescent pseudovirus neutralizing antibody detection method of this application and the traditional Fluc neutralization assay. Figure 16 A shows the correlation between the two methods; Figure 16 B shows the antibody titers for both methods.

[0105] Figure 17 This shows the results of detecting immune serum using an in vitro neutralizing antibody detection method for influenza pseudoviruses.

[0106] Sequence information

[0107] Information on some sequences involved in this invention is shown in Table 1.

[0108] Table 1. Main sequence information Detailed Implementation

[0109] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0110] Unless otherwise specified, the experiments and methods described in the embodiments are performed in accordance with conventional methods well known in the art and described in various references.

[0111] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0112] Example 1. Main Experimental Materials

[0113] 1.1 Experimental plasmids

[0114] (1) Eukaryotic expression vector pSG3Δenv: stored in the AIDS and STD virus vaccine laboratory of the China National Institutes for Food and Drug Control, used to construct various backbone plasmids carrying fluorescent proteins.

[0115] (2) pAmCyan1-C1 Vector: purchased from Clontech, catalog number 632441, used to construct the cyan backbone plasmid SG3-AmCyan.

[0116] (3) pZsGreen1-C1 Vector: purchased from Clontech, catalog number 632447, used to construct the green backbone plasmid SG3-ZsGreen.

[0117] (4) pZsYellow1-C1 Vector: purchased from Clontech, catalog number 632444, used to construct the yellow backbone plasmid SG3-ZsYellow.

[0118] (5) pmOrange2 Vector: Purchased from Clontech, catalog number 632548, used to construct the backbone plasmid SG3-mOrange carrying orange color.

[0119] (6) pE2-Crimson Vector: purchased from Clontech, catalog number 632553, used to construct the SG3-E2 Crimson backbone plasmid carrying a deep red color.

[0120] (7) pdTomato Vector: Purchased from Clontech, catalog number 632531, used to construct the backbone plasmid SG3-tdTomato carrying tomato red color.

[0121] (8) pmCherry Vector: Purchased from Clontech, catalog number 632522, used to construct the backbone plasmid SG3-mCherry carrying cherry red color.

[0122] (9) HA protein plasmid: The nucleotide sequences of the HA proteins of all vaccine strains were synthesized by General Biotechnology Co., Ltd. (Anhui) after mammalian codon optimization, and cloned into the pcDNA3.1(+) vector to obtain the pcDNA3.1-HA plasmid. The amino acid sequences of H1, H3, BV and BY proteins are shown in SEQ ID NO: 24~27, respectively.

[0123] 1.2 Clinical and guinea pig immune sera used in experiments

[0124] (1) The 2020 influenza vaccine-specific antiserum anti-H1, anti-H3, anti-BV, and anti-BY were provided by NIBSC. The 2020 influenza vaccine strains are as follows: A / Guangdong-Maonan / SWL1536 / 2019(H1N1)pdm01, A / HongKong / 2671 / 2019 (H3N2), B / Washington / 02 / 2019 (B / Victorialineage) and B / Phuket / 3073 / 2013 (B / Yamagata lineage).

[0125] (2) Guinea pig immune serum was obtained by immunization with the DNA plasmid of the 2020 influenza vaccine strain of influenza virus.

[0126] (3) Serum from the 2020 influenza vaccine clinical immunization was provided by Beijing Sinovac Biotech Co., Ltd.

[0127] 1.3 Experimental Cells

[0128] (1) HEK-293T: human embryonic kidney cells, preserved in our laboratory, cultured under DMEM (containing 10% FBS).

[0129] (2) Huh 7: human liver cancer cells, preserved in our laboratory, cultured under DMEM (containing 10% FBS).

[0130] (3) Vero: African green monkey kidney cells, preserved in our laboratory, cultured under DMEM (containing 10% FBS).

[0131] (4) MDCK: Canine kidney cells, provided by the cell laboratory, cultured under DMEM (containing 10% FBS).

[0132] (5) Tzmbl: HeLa cells that can stably express HIV-1 receptor and co-receptor, stored in our laboratory, cultured under DMEM (containing 10% FBS).

[0133] (6) AF: Human embryonic kidney cells stably transfected with ACE2 and Fusion receptors were stored in our laboratory and cultured under DMEM (containing 10% FBS).

[0134] 1.4 Main Reagents and Consumables

[0135] Table 2-1 Main Reagents and Consumables

[0137] 1.5 Main Instruments

[0138] Table 2-2 Main Instruments

[0140] 1.6 Commonly Used Solutions

[0141] (1) LB liquid culture medium: Weigh 10g tryptone, 10g NaCl and 5g yeast powder, add to 1L of purified water, and autoclave at 121℃ for 30min.

[0142] (2) LB solid culture medium: Weigh 5g yeast powder, 10g tryptone, 10g NaCl, 12g agar powder, add to 1L of purified water, and autoclave at 121℃ for 30min.

[0143] (3) 1000× Ampicillin (Amp): Prepare a stock solution with a concentration of 100 mg / mL using ddH2O, filter it through a 0.2 μm filter, and store it at -20℃.

[0144] (4) 1000× kanamycin (Kana): Prepare a stock solution with a concentration of 100 mg / mL using ddH2O, filter it through a 0.2 μm filter, and store it at -20℃.

[0145] (5) DMEM complete medium: Add 50-60 mL fetal bovine serum, 5-6 mL penicillin and streptomycin (100 IU / mL each) and 5-6 mL HEPES buffer to 500 mL of DMEM basal medium and store at 4℃.

[0146] (6) 1% DMEM complete medium: Add 5-6 mL fetal bovine serum, 5-6 mL penicillin and streptomycin (100 IU / mL each) and 5-6 mL HEPES buffer to 500 mL of DMEM basal medium, and store at 4℃.

[0147] (7) 50×TAE buffer: Tris 242g, glacial acetic acid 57.1mL, EDTA (pH=8.0) 0.5mol / L 100mL, add water to make up to 1L, and let stand at room temperature.

[0148] (8) Cell cryopreservation solution: Take 9 mL of inactivated fetal bovine serum in 10 mL units and add 1 mL of DMSO and mix well.

[0149] (9) 5-fold dilution of trypsin: Add 20 mL of the original trypsin to 80 mL of PBS buffer to dilute 5 times in 100 mL units, store at 4°C for later use, and equilibrate to room temperature before use.

[0150] (10) Fluorescent detection substrate: Add 250mL buffer to Bright-Glo™ fluorescent detection substrate dry powder, mix by inverting, then add the same volume of PBS buffer to dilute by one-time, aliquot and store in a -20℃ refrigerator protected from light. Thaw at room temperature in the dark before use.

[0151] (11) 1 mol / L NaCHO3: Weigh 84.000 g of NaCHO3, dissolve it in 1 L ddH2O, make up to volume, shake well, filter with a 0.2 μm filter, and store at 4 °C.

[0152] (12) 1 mol / L TPCK: Take 41.6 μL of hydrochloric acid (HCl) and add it to 500 mL of ddH2O to dissolve the TPCK powder to obtain 1 mol / L TPCK.

[0153] (13) Exogenous NA: Dissolve the powder in 7.2 mL of PBS buffer in 25 units to prepare 7 MU / mL exogenous NA.

[0154] 1.7 Laboratory Animals

[0155] The guinea pigs used in this experiment were Hartley guinea pigs, weighing approximately 200g, SPF grade. They were purchased from the Laboratory Animal Resources Institute of the China National Institutes for Food and Drug Control and were housed in the animal laboratory of the China National Institutes for Food and Drug Control.

[0156] Example 2. Construction of influenza virus based on fluorescent pseudovirus

[0157] 2.1 Construction of fluorescent backbone plasmids

[0158] Based on the sequences of fluorescent fragments in seven plasmids—pAmCyan1-C1, pZsGreen1-C1, pZsYellow1-C1, pmOrange2, pE2-Crimson, pdTomato, and pmCherry—eleven pairs of primers were designed to amplify fragments: CMV+AmCyan (nucleotide sequence encoding AmCyan protein containing the CMV promoter, primer sequences shown in SEQ ID NO: 1 and 2), CMV+ZsGreen (nucleotide sequence encoding ZsGreen protein containing the CMV promoter, primer sequences shown in SEQ ID NO: 17 and 18), CMV+ZsYellow (nucleotide sequence encoding ZsYellow protein containing the CMV promoter, primer sequences shown in SEQ ID NO: 3 and 4), mOrange (nucleotide sequence encoding mOrange protein, primer sequences shown in SEQ ID NO: 21 and 22), and E2 Crimson (nucleotide sequence encoding E2 Crimson protein, primer sequences shown in SEQ ID NO: 21 and 22). The sequence includes (as shown in SEQ ID NO: 7 and 8), pdTomato (nucleotide sequence encoding pdTomato protein, primer sequences as shown in SEQ ID NO: 15 and 16), mCherry (nucleotide sequence encoding mCherry protein, primer sequences as shown in SEQ ID NO: 11 and 12), and fragment CMV.

[0159] The nucleotide sequence encoding the AmCyan protein containing the CMV promoter was cloned into the PSG3Δenv vector to construct the recombinant expression plasmid SG3-AmCyan. Similarly, the nucleotide sequence encoding the ZsGreen protein containing the CMV promoter was cloned into the PSG3Δenv vector to construct the recombinant expression plasmid SG3-ZsGreen. The nucleotide sequence encoding the ZsYellow protein containing the CMV promoter was cloned into the PSG3Δenv vector to construct the recombinant expression plasmid SG3-ZsYellow. The nucleotide sequence clone product encoding the mOrange protein was ligated with the nucleotide sequence clone product encoding the CMV protein (primer sequences are shown in SEQ ID NO: 19 and 20), and then cloned into the PSG3Δenv vector to construct the recombinant expression plasmid SG3-mOrange. Similarly, the nucleotide sequence clone product encoding the E2 Crimson protein was ligated with the nucleotide sequence clone product encoding the CMV protein (primer sequences are shown in SEQ ID NO: 5 and 6), and then cloned into the PSG3Δenv vector to construct the recombinant expression plasmid SG3-E2Crimson. The nucleotide sequence clones encoding the pdTomato protein and the CMV protein were ligated together (primer sequences shown in SEQ ID NO: 13 and 14), and then cloned into the PSG3Δenv vector to construct the recombinant expression plasmid SG3-pdTomato. The nucleotide sequence clones encoding the mCherry protein and the CMV protein were ligated together (primer sequences shown in SEQ ID NO: 9 and 10), and then cloned into the PSG3Δenv vector to construct the recombinant expression plasmid SG3-mCherry.

[0160] The amino acid sequences of the fluorescent proteins mCherry, ZsYellow, and AmCyan are shown in SEQ ID NO: 31-33, respectively. The size of each clone fragment is as follows: CMV+AmCyan (1299 bp, SEQ ID NO: 30), CMV+ZsGreen (1308 bp), CMV+ZsYellow (1305 bp, SEQ ID NO: 29), CMV (589 bp), pdTomato (1428 bp), E2 Crimson (678 bp), mCherry (708 bp), and mOrange (719 bp). Figure 1 It can be seen that the nucleotide sequence clones of each fragment are of the correct size, which ensures the successful construction of the fluorescent backbone plasmid.

[0161] Subsequently, the backbone plasmid pSG3Δenv was digested with HpaI. The recovered fluorescent target fragment, identified by agarose gel electrophoresis, was then ligated to the digested backbone vector fragment at a mass ratio of 1:3 using the Clontech In-Fusion® HD Cloning plus kit. The structural diagrams of the obtained fluorescent backbone plasmids are shown below. Figure 2 As shown.

[0162] 2.2 Identification of fluorescent backbone plasmids

[0163] (1) After the fluorescent backbone plasmid was constructed, it was sent to the company for sequencing, and the results were correct.

[0164] (2) In addition, the fluorescent backbone plasmids were transfected into 293T cells separately. After 24 hours, their luminescence was observed under a fluorescence microscope. The results showed that all 7 constructed fluorescent backbone plasmids could emit fluorescence under the corresponding excitation light, so the construction was correct.

[0165] 2.3 Preparation of fluorescent pseudoviruses

[0166] Take the preparation of the pseudovirus H1-AnCyan as an example.

[0167] (1) One day in advance, the fully grown HEK 293T cells (hereinafter referred to as 293T cells) were passaged at a ratio of one to three and seeded into 12-well plates for transfection.

[0168] (2) Preparation tube 1: Take 65 μL opti-MEM into an EP tube, add 2.25 μg of fluorescent backbone plasmid SG3-AmCyan and 0.75 μg of H1 subtype influenza membrane protein expression plasmid HA, mix well and let stand for 5 min.

[0169] (3) Preparation tube 2: Take 65 μL opti-MEM into an EP tube, add Lipofiter transfection reagent, mix well and let stand for 5 min.

[0170] (4) Mix the solutions in tube 1 and tube 2, gently mix and let stand for 20 minutes.

[0171] (5) Add the above solution to the pre-coated 293T cells (the cell density is best at 80%-90%) and mix gently. After culturing the cells in a 5% CO2, 37℃ incubator for 4-8 hours, remove the original culture medium from the culture flask, add 1 mL of fresh 1% DMEM culture medium, and continue culturing for 40 hours.

[0172] (6) After 40 hours, the supernatant was aspirated, centrifuged at 1000g for 10 minutes, filtered through a 0.45μm filter, dispensed, and frozen at -80℃.

[0173] 2.4 Titration of fluorescent pseudoviruses

[0174] (1) Packaging results of different fluorescent backbone plasmids and different subtype influenza virus membrane plasmids

[0175] Plasmids H1, H3, BV, and BY from the 2020 influenza vaccine strains were co-transfected with seven pre-constructed fluorescent backbone plasmids into 293T cells to prepare pseudoviruses, which were then titrated.

[0176] In this embodiment, the preparation of pseudovirus, taking the pseudovirus H1-AmCyan as an example, involved using 2.25 μg of the fluorescent backbone plasmid SG3-AmCyan and 0.75 μg of the H1 subtype influenza membrane protein expression plasmid HA, and transfecting 293T cells using Lipofiter transfection reagent. Upon harvest, the supernatant was aspirated, centrifuged at 1000g for 10 min, filtered through a 0.45 μm filter to obtain the virus stock solution, which was then aliquoted and frozen at -80°C.

[0177] The specific steps of titration are as follows:

[0178] (1) Before the experiment, the DMEM complete culture medium was equilibrated to room temperature, and the pseudovirus that had been frozen to -80℃ was thawed in a water bath at room temperature.

[0179] (2) Take a sterile 96-well cell culture plate and add 100 μL of DMEM complete culture medium to each well.

[0180] (3) Add 50 μL of the first pseudovirus H1-AmCyan to wells B2-B3 in row 1, 50 μL of the second pseudovirus H3-Cherry to wells B4-B5 in row 1, 50 μL of the third pseudovirus BV-Yellow to wells B6-B7 in row 1, and 50 μL of the fourth pseudovirus BY-AmCyan to wells B8-B9 in row 1. Wells B10-B11 are left untreated and used as cell controls. The sample layout is shown in Table 3-1.

[0181] (4) Adjust the multichannel pipette to 50 μL, gently blow and aspirate the liquid in the first row well 6 to 8 times to mix it thoroughly, then transfer 50 μL of liquid to the corresponding second row, and repeat this process to dilute to the sixth row, and then discard 50 μL of liquid.

[0182] (5) Preparation containing 5×10 5 MDCK cell suspension at 100 cells / mL: MDCK cells were digested, followed by cell counting, and then diluted to 5 × 10⁶ cells / mL with DMEM complete medium. 5 per mL.

[0183] (6) Add cell suspension: Add 100 μL of 5×10⁻⁶ mcg ...5 DMEM containing MDCK cells / mL was applied to each well to achieve a cell density of 5 × 10⁶ cells / mL. 4 indivual.

[0184] (7) Cell culture: Place in a cell culture incubator at 37℃ and 5% CO2 and culture for 96 h.

[0185] (8) Plate reading: After 96 hours, remove the 96-well plate from the cell culture incubator and read the number of fluorescent spots using BioTek. The experimental results are shown in Table 3-2.

[0186] Table 3-1 Sample Layout for Pseudovirus Titration

[0187]

[0188] Note: CC refers to cell control wells, containing only DMEM complete medium and cells.

[0189] Table 3-2 Packaging of HA membrane plasmids and different backbone plasmids for different influenza subtypes

[0190]

[0191] Note: The numbers in the table represent the pseudovirus stock solution diluted 3-fold in the titration experiment, i.e., the number of fluorescent spots after sample 1 infected MDCK cells.

[0192] As shown in Table 3-2, the pseudoviruses formed by packaging the fluorescent backbone plasmids SG3-mOrange and SG3-E2 Crimson with the HA membrane protein particles of different influenza subtypes exhibited low fluorescence counts, or even almost no fluorescence, after infecting cells. This indicates low infection efficiency and poor packaging effect of the pseudoviruses, hence these two fluorescent backbone plasmids were discarded. However, the backbone plasmid SG3-ZsYellow, after packaging with four subtype membrane protein particles, showed high fluorescence counts in titration experiments. In particular, the pseudoviruses formed after packaging with the BV membrane protein particle showed significantly higher fluorescence counts than other pseudoviruses. This demonstrates the high stability of the SG3-ZsYellow backbone plasmid genome and its high compatibility with various membrane protein particles. It not only achieves high assembly efficiency, correct membrane protein integration, and normal expression of fluorescent genes, but also produces pseudoviruses with high infectivity. For ease of detection, the three fluorescent backbone plasmids SG3-AmCyan, SG3-ZsYellow, and SG3-mCherry were selected to package pseudoviruses with the influenza virus membrane protein particle HA. Based on the packaging results of the three fluorescent backbones (SG3-AmCyan, SG3-ZsYellow, and SG3-mCherry) and the four subtypes (H1, H3, BV, and BY), it was determined that SG3-AmCyan packaged pseudoviruses with HA membrane protein particles of influenza H1 and BY subtypes, SG3-ZsYellow with HA membrane protein particles of BV subtype, and SG3-mCherry with HA membrane protein particles of H3 subtype. In other words, four types of pseudoviruses—H1-AmCyan, BY-AmCyan, BV-Yellow, and H3-Cherry—were obtained for further research, and will be referred to hereafter as H1, BY, BV, and H3, respectively.

[0193] 2.5 Optimization of the fake virus packaging system

[0194] (1) Selection of enzyme digestion reagents

[0195] After harvesting the pseudovirus, it was treated with either EDTA trypsin (50 μL) or TPCK trypsin (20 μL), followed by titration. Untreated pseudovirus was used as a control, and the fluorescence results were observed. Figure 3 It can be seen that the number of fluorescent spots in the four types of pseudoviruses treated with TPCK trypsin was 2-4 times that of those treated with EDTA trypsin, while untreated pseudoviruses could not infect cells. Therefore, TPCK trypsin treatment is the optimal method after harvesting pseudoviruses.

[0196] (2) Selection of enzyme digestion reagent concentration

[0197] After determining that TPCK trypsin should be used to treat the pseudovirus, the concentration of TPCK trypsin was further determined. The harvested pseudoviruses were treated with TPCK at concentrations of 10 µg / ml, 20 µg / ml, 40 µg / ml, 80 µg / ml, 160 µg / ml, and 320 µg / ml, respectively, followed by titration, and the fluorescence results were observed.

[0198] from Figure 4 It can be seen that for H1 and BY subtype pseudoviruses, the number of fluorescent spots was very low after treatment with TPCK concentrations of 10 µg / ml and 20 µg / ml. When the TPCK concentration was increased to 40 µg / ml, the number of fluorescent spots increased significantly. Further increases in TPCK concentration to 80 µg / ml also resulted in a corresponding increase in the number of fluorescent spots, but subsequent increases in TPCK concentration did not significantly change the number of fluorescent spots. For H3 and BV subtype pseudoviruses, approximately 500 fluorescent spots were observed after treatment with TPCK concentrations of 10 µg / ml and 20 µg / ml. When the concentration was increased to 40 µg / ml, approximately 1000 fluorescent spots were observed. Further increases in TPCK concentration did not increase the number of fluorescent spots for H3 subtype pseudoviruses, while the number of fluorescent spots for BV subtype pseudoviruses decreased significantly. Therefore, it is optimal to treat the harvested H1 and BY subtype pseudoviruses with 80 µg / ml TPCK and the harvested H3 and BV subtype pseudoviruses with 40 µg / ml TPCK.

[0199] (3) Selection of transfection ratio

[0200] After determining the enzyme digestion reagents and concentrations, the co-transfection ratio of the fluorescent backbone plasmid SG3 and the influenza membrane protein plasmid HA was optimized. Transfection was performed at backbone plasmid to membrane protein plasmid mass ratios of 3:1, 2:1, 1:1, 1:2, and 1:3, and the harvested pseudoviruses were titrated. Figure 5 The results showed that the highest number of fluorescent spots in the pseudoviruses were obtained when the ratio of backbone plasmid to membrane plasmid transfection was 3:1. Therefore, the optimal ratio of backbone plasmid to membrane protein expression plasmid for pseudovirus packaging was determined to be 3:1.

[0201] Example 3. Establishment of a method for detecting influenza virus neutralizing antibodies based on fluorescent pseudoviruses

[0202] Influenza pseudoviruses were prepared according to the optimized pseudovirus packaging system, centrifuged, filtered, and aliquoted (and stored at -80℃) for subsequent methodological studies. This experiment investigated cell tropism, cell inoculum size, detection time, and virus addition to determine optimal detection conditions and establish a stable and reliable in vitro neutralizing antibody detection method for influenza virus based on fluorescent pseudoviruses.

[0203] 3.1 Selection of Sensitive Cells

[0204] Cellular tropism of pseudoviruses is a crucial indicator of the stability of antibody detection methods, ensuring maximum experimental stability. 293T, MDCK, Vero, AF, and TZmbl cells were selected as candidate cells. Diluted pseudoviruses were added to 96-well plates, and the fluorescence count was measured 72 hours after infection with each of the five cell types. Results are shown below. Figure 6 As shown, the relative number of fluorescent spots in MDCK cells infected with influenza pseudovirus was the highest and the consistency was good. Therefore, MDCK cells were selected as the cells for subsequent in vitro neutralizing antibody detection methods.

[0205] 3.2 Determination of Cell Addition Amount

[0206] After determining the sensitive cells for the in vitro neutralizing antibody detection method, the cell seeding amount was then selected. Diluted influenza pseudoviruses H1, H3, BV, and BY were added to 96-well plates, along with different numbers of MDCK cells: 5000 cells / well, 10000 cells / well, 20000 cells / well, 30000 cells / well, 40000 cells / well, 50000 cells / well, and 60000 cells / well. Fluorescent spots were counted 72 hours after infection. The results showed ( Figure 7 As the cell seeding density increases, the number of fluorescent spots gradually increases, reaching its maximum at a seeding density of 50,000 cells / well. Further increases in cell number thereafter reduce the observed number of fluorescent spots. Therefore, 50,000 cells / well was chosen as the seeding density for the in vitro neutralizing antibody detection method.

[0207] 3.3 Determination of Detection Time

[0208] Diluted influenza pseudovirus was added to 96-well plates, followed by 50,000 MDCK cells per well. Fluorescence counts were measured at 24 h, 48 h, 72 h, 96 h, 120 h, and 144 h to determine the optimal detection time for the in vitro neutralizing antibody assay. Results are as follows: Figure 8 As shown in the figure. Comparison revealed that no fluorescence was observed at 24 h due to the short infection time of the MDCK pseudovirus; the number of fluorescent spots increased with time, but there was no significant increase after 96 h. Therefore, in in vitro neutralizing antibody detection, fluorescence detection was performed 96 h after influenza pseudovirus infection of cells.

[0209] 3.4 Determination of the amount of fake virus added

[0210] After determining the infected cells, cell number, and detection time for the in vitro neutralization experiment, we further optimized the pseudovirus inoculation amount for the neutralization experiment. Clinically immunized serum was serially diluted in 96-well plates, and then 1 TCID₂ was added to each well. 505TCID 50 10TCID 50 15TCID 50 20TCID 50 30TCID 50 The fake viruses H1-AmCyAn, H3-Cherry, BV-Yellow, and BY-AmCyan. (The text appears to be incomplete and contains several errors. A more accurate translation would require the full context.) Figure 9 It can be seen that for the H1-AmCyan pseudovirus, adding 1TCID... 50 At that time, the inhibition curve of the neutralization experiment was not smooth, so 5 TCID was added. 50 -20TCID 50 At that time, the inhibition curves of the neutralization experiment showed no significant difference, but after adding 30 TCID... 50 At that time, the detection sensitivity decreases. For H3-Cherry pseudoviruses, adding 1 TCID... 50 At that time, the inhibition curve of the neutralization experiment showed good smoothness and high sensitivity, with the addition of 5 TCID. 50 -30TCID 50 At that time, the detection sensitivity decreased significantly. For the BV-Yellow and BY-amCyan pseudoviruses, adding 1 TCID... 50 At that time, the inhibition curve of the neutralization experiment showed good smoothness and high sensitivity. In summary, the amount of pseudovirus added in in vitro neutralizing antibody detection should be controlled at 1 TCID. 50 -5TCID 50 .

[0211] Example 4. Validation of the influenza virus neutralizing antibody detection method based on fluorescent pseudoviruses

[0212] 4.1 Sensitivity

[0213] Antiserum for H1 subtype was serially diluted 150, 750, 3750, 18750, 93750, and 281250 times, and then incubated with H1-AmCyan pseudovirus; antiserum for H3 subtype was serially diluted 150, 750, 3750, 18750, 93750, and 281250 times, and then incubated with H3-Cherry pseudovirus; antiserum for BV subtype was serially diluted 150, 750, 3750, 18750, 93750, and 281250 times, and then incubated with BV-Yellow pseudovirus; antiserum for BY subtype was serially diluted 150, 750, 3750, 18750, 93750, and 281250 times, and then incubated with BY-AmCyan pseudovirus. Antisera against four different virus types were tested using four pseudoviruses: H1-AmCyan, H3-Cherry, BV-Yellow, and BY-AmCyan. The results are as follows: Figure 10 As shown, H1-AmCyan detects the ID of isotype antibodies. 50 For 19621, the ID of H3-Cherry for detecting isotype antibodies 50 The value is 5695, and the ID of the BV-Yellow isotype antibody is detected. 50 The ID of the BY-AmCyan is 10936, used to detect isotype antibodies. 50 The value was 2793. The four subtypes of pseudoviruses, H1-AmCyan, H3-Cherry, BV-Yellow, and BY-AmCyan, all showed high sensitivity in detecting serum antibodies.

[0214] 4.2 Specificity

[0215] Current influenza vaccines are trivalent or quadrivalent. If several types of neutralizing antibodies could be detected simultaneously, the throughput of pseudovirus neutralizing antibody detection methods could be increased exponentially. To enable the detection of different pseudovirus subtypes in the same serum sample, there must be no cross-protection between subtypes; a single subtype of pseudovirus should only neutralize antibodies corresponding to that subtype and not antibodies from other subtypes. To verify the specificity of the pseudovirus proposed in this application, the following experiments were conducted.

[0216] Antisera from the four subtypes H1, H3, BV, and BY were serially diluted 150, 750, 3750, 18750, 93750, and 281250 times, and then incubated with pseudoviruses from the four subtypes H1-AmCyan, H3-Cherry, BV-Yellow, and BY-AmCyan. Figure 11It can be seen that the anti-H1 subtype serum only neutralizes the H1-AmCyan pseudovirus of the same subtype, and has no neutralization effect on the other three pseudoviruses H3-Cherry, BV-Yellow, and BY-AmCyan. The anti-H3 subtype serum only neutralizes the H3-Cherry pseudovirus of the same subtype, and has no neutralization effect on the other three pseudoviruses H1-AmCyan, BV-Yellow, and BY-AmCyan. The anti-BV subtype serum only neutralizes the BV-Yellow pseudovirus of the same subtype, and has no neutralization effect on the other three pseudoviruses H1-AmCyan, H3-Cherry, and BY-AmCyan. The anti-BY subtype serum only neutralizes the BY-AmCyan pseudovirus of the same subtype, and has no neutralization effect on the other three pseudoviruses H1-AmCyan, H3-Cherry, and BV-Yellow. Therefore, the use of the four pseudoviruses H1-AmCyan, H3-Cherry, BV-Yellow, and BY-AmCyan for in vitro neutralizing antibody detection has good specificity and will not be neutralized by neutralizing antibodies from other pseudoviruses.

[0217] 4.3 Detection of Single Subtype Pseudoviruses with Single Subtype Antiserum and Mixed Antiserum

[0218] The serum produced after clinical vaccine immunization is all multivalent serum. In order to detect whether mixed serum has any impact on the detection results of single subtype pseudovirus, the following experiment was conducted.

[0219] Single-subtype antisera were prepared: anti H1, anti H3, anti BV, and anti BY. Equal volumes of serum were then taken from each subtype and mixed to obtain a mixed antiserum: anti H1+H3+BV+BY. Anti H1 and anti H1+H3+BV+BY were serially diluted simultaneously in the same 96-well plate. After incubation with diluted H1-AmCyan pseudovirus, the fluorescence count was detected after 96 hours. Similarly, anti H3 and anti H1+H3+BV+BY were serially diluted simultaneously in the same 96-well plate. After incubation with diluted H3-Cherry pseudovirus, the fluorescence count was detected after 96 hours. Finally, anti BV and anti H1+H3+BV+BY were serially diluted simultaneously in the same 96-well plate. After incubation with diluted BV-Yellow pseudovirus, the fluorescence count was detected after 96 hours. Anti-BY and anti-H1+H3+BV+BY were serially diluted simultaneously on the same 96-well plate, followed by incubation with diluted BY-AmCyan pseudovirus. Fluorescence spot count was detected after 96 hours. Figure 12It can be seen that the results of H1-AmCyan pseudovirus detection of anti-H1 serum are no different from those of mixed antiserum; the results of H3-Cherry pseudovirus detection of anti-H3 serum are no different from those of mixed antiserum; the results of BV-Yellow pseudovirus detection of anti-BV serum are no different from those of mixed antiserum; and the results of BY-AmCyan pseudovirus detection of anti-BY serum are no different from those of mixed antiserum. Therefore, serum after multivalent vaccine immunization does not affect the neutralization results of single-subtype pseudoviruses. That is, the pseudovirus of this application will not be interfered with when detecting samples containing neutralizing antibodies of multiple subtypes. This indicates that the pseudovirus of this application can accurately and efficiently detect single-subtype neutralizing antibodies in multivalent immunized serum.

[0220] 4.4 Comparison of dual-color and single-color neutralizing antibody detection

[0221] Most influenza vaccines currently on the market or under development are quadrivalent. If several types of neutralizing antibodies could be detected simultaneously, the throughput of pseudovirus neutralizing antibody detection methods could be increased exponentially. We use a 2+2 or 3+1 pseudovirus combination method to detect neutralizing antibodies in serum. The 2+2 method involves testing with two influenza A pseudoviruses combined once, followed by testing with two influenza B pseudoviruses combined once.

[0222] (1) Two combinations of influenza A pseudoviruses H1-AmCyan and H3-Cherry, and two combinations of influenza B pseudoviruses BV-Yellow and BY-AmCyan were used to test serum twice.

[0223] To determine whether mixing a single pseudovirus subtype with another pseudovirus would affect the results of the neutralization assay, eight serum samples were collected after clinical immunization. The serum was diluted in 96-well plates, and then four single-subtype pseudoviruses (H1-AmCyan, H3-Cherry, BV-Yellow, BY-AmCyan, H1-AmCyan+H3-Cherry, and BV-Yellow+BY-AmCyan) and two mixed-subtype pseudoviruses were added. The number of fluorescent spots was detected after 96 hours. The results were compared between testing the same post-clinical immunization serum sample before and after mixing with a single pseudovirus subtype with another. Figure 13As can be seen, the results of testing 8 serum samples with the pseudovirus H1-AmCyan alone were not significantly different from the results of testing the same serum samples with a mixture of pseudovirus H3-Cherry and pseudovirus H3-Cherry. Similarly, the results of testing 8 serum samples with the pseudovirus BV-Yellow alone were not significantly different from the results of testing the same serum samples with a mixture of pseudovirus BY-AmCyan and pseudovirus BY-AmCyan. This further demonstrates the accuracy and feasibility of using the combination of H1-AmCyan and H3-Cherry subviruses to detect multivalent immune sera, as well as the accuracy and feasibility of using the combination of BV-Yellow and BY-AmCyan subviruses to detect multivalent immune sera.

[0224] In summary, there is no significant difference between the results of dual-color and single-color neutralizing antibody detection. Therefore, the 2+2 method is feasible for detecting influenza virus neutralizing antibodies in serum. Compared with single-color neutralizing antibody detection, dual-color neutralizing antibody detection can save at least half of the manpower and resources.

[0225] (2) To investigate the potential of different fluorescent pseudovirus combinations for detection, we experimented with the compatibility of various fluorescent proteins, including nine fluorescent proteins: AmCyan, ZsYellow, E2 Crimson, mCherry, mOrange, pdTomato, EGFP, RFP, and ZsGreen. Furthermore, we constructed different plasmids expressing these nine fluorescent proteins: SG3-AmCyan, SG3-ZsYellow, SG3-E2 Crimson, SG3-mCherry, SG3-mOrange, SG3-pdTomato, SG3-EGFP, SG3-RFP, and SG3-ZsGreen. We then transfected these plasmids individually into 293T cells and used six excitation sources (CFP, YFP, Texas Red, GFP, RFP, and CY5) on a BIOTEK instrument to detect the luminescence of different fluorescent plasmids under different excitation sources. The results showed that SG3-AmCyan and SG3-ZsGreen used the same excitation source. Considering that SG3-ZsGreen had poor miniaturization performance, it was discarded. The luminescence of the remaining 8 fluorescent plasmids is shown in Table 4.

[0226] Table 4. Luminescence of different fluorescent plasmids under different excitation sources

[0227]

[0228] The results showed that signals from different fluorescent proteins may overlap under specific detection channels; that is, their emission spectra overlap, causing signal interference and making it impossible to distinguish which fluorescent protein produced the signal. For example, when SG3-ZsYellow and SG3-mOrange were used together, since SG3-ZsYellow generated signals in the YFP, GFP, and RFP channels, while SG3-mOrange generated signals in the YFP and RFP channels, the signal of SG3-mOrange would overlap with that of SG3-ZsYellow under any excitation light source. In other words, these two fluorescent proteins cannot be used for two-color neutralizing antibody detection. We also observed weak crossover between SG3-E2 Crimson and SG3-mCherry under Texas Red and CY5 excitation light sources, meaning that weak mutual interference signals were detected under Texas Red or CY5 channels.

[0229] Furthermore, we found that the three fluorescence combinations of SG3-AmCyan, SG3-ZsYellow, and SG3-E2 Crimson, or the three fluorescence combinations of SG3-AmCyan, SG3-ZsYellow, and SG3-mCherry, are completely distinct and can be distinguished within the same detection system. This conclusion provides a spectral compatibility basis for designing neutralizing antibody detection experiments using multicolor fluorescent pseudoviruses and also demonstrates the uniqueness of the aforementioned fluorescence combinations.

[0230] 4.5 Comparison of three-color neutralizing antibody detection and single-color neutralizing antibody detection

[0231] Based on the spectral compatibility results of various fluorescent proteins, we constructed a three-color neutralizing antibody detection method according to the packaging efficiency of each fluorescent protein combined with different HA proteins. This method detects neutralizing antibodies against four subtypes (H1, H3, BV, and BY) in two separate tests. The 3+1 combination method for detecting influenza pseudovirus neutralizing antibodies involves detecting a combination of three pseudoviruses: H1-AmCyan, H3-Cherry, and BV-Yellow, with BY-AmCyan detected alone. The same serum sample is tested twice. Furthermore, the prevalence of BY influenza virus has declined in recent years, and trivalent vaccines targeting H1, H3, and BV are expected to become the mainstream in the future. This means that in most scenarios, only neutralizing antibodies against H1, H3, and BV subtypes need to be detected. Therefore, this method using a combination of three pseudoviruses (H1-AmCyan, H3-Cherry, and BV-Yellow) can complete the detection of neutralizing antibodies in serum in a single test.

[0232] To determine whether mixing a single pseudovirus subtype with two other pseudovirus subtypes would affect the results of the neutralization assay, eight serum aliquots from post-clinical immunization were collected. The serum was diluted in 96-well plates, and then three single pseudovirus subtypes (H1-AmCyan, H3-Cherry, and BV-Yellow) and a mixture of the three subtypes (H1-AmCyan + H3-Cherry + BV-Yellow) were added. The number of fluorescent spots was measured after 96 hours. The results were compared between testing the same clinical immunized serum aliquots before and after mixing with the same pseudovirus subtype and with the other two subtypes.

[0233] Depend on Figure 14 It can be seen that the results of detecting 8 serum samples with H1-AmCyan alone are not significantly different from the results of detecting the same serum samples mixed with H3-Cherry and BV-Yellow pseudoviruses. Similarly, the results of detecting 8 serum samples with H3-Cherry alone are not significantly different from the results of detecting the same serum samples mixed with H1-AmCyan and BV-Yellow pseudoviruses. The results of detecting 8 serum samples with BV-Yellow alone are not significantly different from the results of detecting the same serum samples mixed with H1-AmCyan and H3-Cherry pseudoviruses. This demonstrates the accuracy and feasibility of using a combination of H1-AmCyan, H3-Cherry, and BV-Yellow subviruses to detect multivalent immune sera. In summary, there is no significant difference between three-color and single-color neutralizing antibody detection; therefore, the 3+1 model is feasible for detecting influenza virus neutralizing antibodies in serum. Compared with single-color neutralizing antibody detection, three-color neutralizing antibody detection can save at least half of the manpower and resources.

[0234] 4.6 Repeatability

[0235] To analyze the reproducibility of the detection method, six serum samples were collected after clinical immunization. The serum was diluted in 96-well plates, and then a 3+1 pseudovirus detection method was used. H1-AmCyan + H3-Cherry + BV-Yellow pseudovirus was added, and the assay was repeated three times at three different time points, with four replicates per replicate. The number of fluorescent spots was detected after 96 hours. The results are as follows: Figure 15 As shown, the detection method of this application has good repeatability.

[0236] 4.7 Comparison of detection methods for fluorescent protein-labeled pseudoviruses and luciferase (Fluc)-labeled pseudoviruses with neutralizing antibodies

[0237] Sixteen serum samples collected after clinical immunization against influenza were selected, and the levels of neutralizing antibodies in the serum were detected using H1-AmCyan and H1-Fluc pseudoviruses, respectively. Figure 16 (A) It can be seen that the two methods have a good correlation, with a correlation coefficient R.2 The value was 0.85. The geometric mean of neutralizing antibody titers against the influenza H1 subtype was calculated for both methods. It was found that the antibody titer detected by the fluorescent pseudovirus neutralizing antibody test was approximately twice as high as that detected by the traditional Fluc neutralization assay (P < 0.01). Figure 16 B).

[0238] Example 5. Preliminary application of a fluorescent pseudovirus-based method for detecting influenza virus neutralizing antibodies.

[0239] The established 3+1 method for detecting influenza virus neutralizing antibodies based on fluorescent pseudoviruses—H1-AmCyan + H3-Cherry + BV-Yellow and BY-AmCyan—was used to detect the efficacy of the vaccine in two separate assays, involving 64 serum samples, including 53 post-clinical immunization sera and 11 guinea pig immune sera. The vaccine used is described in Example 1.2. Guinea pigs were immunized with a dose of 200 µg per animal via intramuscular injection, followed by three immunizations every 14 days.

[0240] Depend on Figure 17 As shown in Table 5, among the 64 serum samples tested, except for serum samples H29 and T11 which did not produce any of the four antibodies, and sample T5 which did not produce antibodies against the BV subtype, all other serum samples produced antibodies against the four subtypes of the 2020 influenza vaccine strains H1, H3, BV, and BY. Therefore, both clinical vaccine immunization and guinea pig immunization had a protective effect.

[0241] Table 5. Results of Detection of Immune Serum Using In Vitro Neutralizing Antibodies Against Influenza Pseudoviruses

[0242] Note: Samples H1-H43 are serum from clinical immunization, and T1-T11 are guinea pig immunized serum. Values ​​in the table are ID values. 50 .

[0243] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A vector comprising, based on the pSG3Δenv vector, an exogenous sequence as shown in SEQ ID NO: 29, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 29, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to SEQ ID NO: 29; Preferably, the pSG3Δenv vector contains or has a nucleotide sequence as shown in SEQ ID NO: 23, or a nucleotide sequence having at least 80% sequence identity with it; Preferably, the exogenous sequence is inserted in the pSG3Δenv vector at the position between bases 13526 and 13527 of the nucleotide sequence corresponding to SEQ ID NO:

23.

2. A vector combination comprising the vector of claim 1 and an influenza virus HA (Hemagglutinin) protein expression plasmid; Preferably, the influenza virus HA protein expression plasmid contains a nucleotide sequence encoding an H1 protein, an H3 protein, a BV protein, or a BY protein; Preferably, the influenza virus HA protein expression plasmid comprises a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27; Preferably, the influenza virus HA protein expression plasmid contains a nucleotide sequence encoding the amino acid sequence of the BV protein; Preferably, the influenza virus HA protein expression plasmid comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 26; or has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 26, or has a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to SEQ ID NO:

26.

3. A host cell comprising the vector of claim 1 or a combination of the vectors of claim 2; The host cell is a human cell, such as a hematopoietic cell, epithelial cell, hepatocyte, tumor cell, or nerve cell; Preferably, the host cell is a HEK 293T cell.

4. A method for preparing a pseudovirus, comprising: (1) Transfect the vector of claim 1 or the combination of the vectors of claim 2 into the host cell of claim 3; and (2) Harvesting fake viruses; Preferably, the method is performed through the following steps: (a) Providing the vector of claim 1 and the influenza virus HA protein expression plasmid; (b) Transfecting the vector of claim 1 and the influenza virus HA protein expression plasmid into the host cells as described above; and (c) Harvesting fake viruses; Preferably, in step (b), the mass ratio of the vector of claim 1 to the influenza virus HA protein expression plasmid is 4:1 to 1:1 (e.g., 3:1, 2:1, 1:1). Preferably, the method further includes treating the harvested pseudovirus with TPCK trypsin (e.g., 40-80 µg / ml of TPCK trypsin).

5. A pseudovirus prepared by the method of claim 4; Preferably, the pseudovirus has any one of the following characteristics: (1) The pseudovirus comprises a protein having the amino acid sequences shown in SEQ ID NO: 32 and SEQ ID NO: 26; (2) The pseudovirus comprises a protein having the amino acid sequences shown in SEQ ID NO: 32 and SEQ ID NO: 24; (3) The pseudovirus comprises a protein having the amino acid sequences shown in SEQ ID NO: 32 and SEQ ID NO: 25; or (4) The pseudovirus contains a protein having the amino acid sequences shown in SEQ ID NO: 32 and SEQ ID NO:

27.

6. A fake virus combination comprising the fake virus of claim 5, and additional fake viruses; Preferably, the fake virus combination includes: A first pseudovirus comprising a protein having the amino acid sequences shown in SEQ ID NO: 32 and SEQ ID NO: 26; a second pseudovirus comprising a protein having the amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 33; and / or a third pseudovirus comprising a protein having the amino acid sequences shown in SEQ ID NO: 25 and SEQ ID NO:

31.

7. A method for detecting the neutralizing activity of influenza virus antibodies in a sample, the method comprising contacting the sample with the pseudovirus or pseudovirus combination before, simultaneously with, or after contacting the pseudovirus of claim 5 or the pseudovirus combination of claim 6 with a host cell; Preferably, the method includes: (1) The pseudovirus of claim 5 or the combination of pseudoviruses of claim 6 is brought into contact with the sample; (2) Introduce the pseudovirus or pseudovirus combination obtained in (1) into the host cell; (3) Under conditions where fluorescence is emitted, observe the host cells, count the number of positive cells in the host cells, and calculate the ID. 50 The value was used to detect the neutralizing activity of the influenza virus antibodies.

8. The method of claim 7, wherein, The method is performed through the following steps: (a) The first fake virus, the second fake virus and the third fake virus are brought into contact with the same sample (e.g., the first fake virus, the second fake virus and the third fake virus are brought into contact with the same sample simultaneously). (b) Take the sample from step (a) and bring it into contact with the host cells; (c) Under conditions where the fluorescence of the first pseudovirus, the second pseudovirus, and the third pseudovirus can be detected, the host cells are observed, the number of positive cells in the host cells is counted, and the ID is calculated. 50 The value was used to detect the neutralizing activity of the influenza virus antibodies; Preferably, the host cell is a mammalian cell, such as human cells, dog cells, or monkey cells; Preferably, the host cell is an MDCK cell; Preferably, the sample is selected from plasma, serum, or any combination thereof; Preferably, the sample contains influenza virus antibodies against multiple subtypes of influenza virus (e.g., anti-H1 subtype antibody, anti-H3 subtype antibody and / or anti-BV subtype antibody).

9. A method for screening candidate drugs capable of inhibiting influenza virus infection of cells, the method comprising contacting the candidate drug with the pseudovirus or pseudovirus combination before, simultaneously with, or after contacting the pseudovirus of claim 5 or the pseudovirus combination of claim 6 with the host cell; Preferably, the method includes: (1) Contact the pseudovirus of claim 5 or the combination of pseudoviruses of claim 6 with the candidate drug; (2) Introduce the pseudovirus or pseudovirus combination obtained in (1) into the host cell; (3) Under conditions where fluorescence is emitted, observe the host cells to determine whether the candidate drug can inhibit influenza virus infection of cells.

10. The method of claim 9, wherein, The method is performed through the following steps: (a) Contact the first pseudovirus, the second pseudovirus, and the third pseudovirus with the candidate drug; (b) Take the sample from step (a) and bring it into contact with the host cells; (c) Under conditions where the fluorescence of the first pseudovirus, the second pseudovirus, and the third pseudovirus can be detected, the host cells are observed, the number of positive cells in the host cells is counted, and the ID is calculated. 50 The value was used to evaluate the drug's ability to inhibit influenza virus infection of cells; Preferably, the host cell is a mammalian cell, such as human cells, dog cells, or monkey cells; Preferably, the host cell is an MDCK cell.

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