CN107868121B

CN107868121B - H3 subtype influenza virus hemagglutinin specific conserved epitope, antibody thereof and application thereof

Info

Publication number: CN107868121B
Application number: CN201610862674.1A
Authority: CN
Inventors: 王健伟; 郭丽; 周红莉
Original assignee: Institute of Pathogen Biology of CAMS
Current assignee: Institute of Pathogen Biology of CAMS
Priority date: 2016-09-28
Filing date: 2016-09-28
Publication date: 2021-03-16
Anticipated expiration: 2036-09-28
Also published as: CN107868121A

Abstract

The invention relates to a hemagglutinin specific conserved epitope of an H3 subtype influenza virus, an antibody specifically binding to the epitope and application of the epitope in diagnosing H3N2 virus infection.

Description

H3 subtype influenza virus hemagglutinin specific conserved epitope, antibody thereof and application thereof

Technical Field

Background

Influenza virus is the main pathogen causing acute upper respiratory tract infection, and infected persons have high morbidity and mortality. According to WHO statistics, an annual influenza epidemic causes about 300-500 million people to cause severe disease, of which about 25-50 million people die (see: WHO. fact sheet Number 211. infiluenza (Seasonal). The annual influenza disease burden is due in part to two co-circulating influenza a viruses-H1N 1 and H3N 2-. An investigation by 1979-2001 in the United states has shown that H3N2 is the most prominent influenza subtype responsible for the morbidity and mortality of influenza viruses, followed by H1N1 and influenza B viruses (see Thompson WW, et al, influenza-associated pathologies in the United states. JAMA,2004,292: 1333-40).

Influenza viruses evolve rapidly, adapt to new hosts, and evade the ability of Host immunity, so that they can continue to pandemic among humans (Taubenberger JK, et al. Influenza virus HAs two glycoproteins on its surface, Hemagglutinin (HA) and Neuraminidase (NA), which are the main targets of the host immune response to influenza virus. Host proteases cleave HA into two subunits HA1 and HA2, HA1 binds to host receptors, HA2 is responsible for binding of the viral envelope to the membrane of host endocytoses (Krystal M, et al. evolution of influenza A and B viruses: conservation of structural defects in the hemagglutinin genes. Proc Natl Acad Sci U S A,1982,79: 4800-4). Changes in the amino acids of the HA1 subunit will interfere with virus recognition by host antibodies, while the HA2 subunit is relatively conserved among the HA subgroup.

The H3N2 influenza virus is one of the viruses responsible for seasonal influenza, causing severe disease and death in high risk groups. Studies have shown that cross-reactive antibodies against different strains and subtypes of influenza virus are produced after infection of the body with influenza virus (Noisumdaeng P, et al. Homosubtypic and heterosubtypic antibodies against high strain and pathogenic fungi inflenza H5N1 recombinant proteins in H5N1 subvors and non-H5N1 subvectors. virology,2014, 454-puzzling 455254-62). Furthermore, there is also a two-way cross-reaction between H3N2 and H7N9 viruses (Guo L, et al. Cross-reactivity beta antigen influenza A (H7N9) virus and diggent H7 subtype-and heterologous influenza A viruses. Sci Rep,2016,622045). Different subtypes of influenza viruses, such as seasonal influenza and avian influenza, can lead to different clinical outcomes for patients. Therefore, diagnosis of H3N2 influenza virus infection should take into account cross-reactivity between different subtypes of influenza virus, and a specific diagnosis method for H3N2 influenza virus infection should be developed. However, the continuous evolution of H3N2 influenza viruses has made it particularly important to identify conserved epitopes specific for the H3 subtype for specific diagnosis of H3N2 influenza viruses.

In this study, we succeeded in identifying two specific epitopes that are highly conserved in H3N 2. On the basis, an H3N2 virus immunofluorescence detection method and an IgG ELISA detection method based on specific epitopes are established, and the IgG ELISA detection method and the hemagglutination inhibition detection method have good consistency.

Disclosure of Invention

In the research, the synthetic peptide library is used for screening the antibody of the H3HA protein to obtain two antigen epitopes with stronger reactivity. The antigen epitope polypeptide is coupled with KLH and then immunized with mice to obtain antiserum. The two epitopes are determined to be specific epitopes highly conserved in H3 subtype influenza virus strains of different years by enzyme-linked immunosorbent assay, immunoblotting identification, sequence alignment and other methods. Based on the epitope, an H3N2 virus immunofluorescence detection method and an IgG ELISA detection method based on the specific epitope are established.

In a first aspect, the invention provides a specific epitope of hemagglutinin of an influenza virus subtype H3, which has the amino acid sequence ICDSPHQILDGKNCT.

In a second aspect, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds to an epitope as defined in the first aspect, wherein the antibody is preferably an IgG antibody.

In a third aspect, the invention provides the antibody or antigen-binding fragment of the second aspect which is a monoclonal antibody or antigen-binding fragment thereof.

In a fourth aspect, the invention provides a method of preparing an immune serum comprising an antibody or antigen-binding fragment of the second aspect, said method comprising immunizing an animal with a synthetic conjugate of an epitope of the first aspect and a carrier, preferably KLH, and collecting blood and isolating serum after immunization.

In a fifth aspect, the invention provides the use of an antibody or antigen-binding fragment of the second or third aspect in the preparation of a reagent for detecting an influenza H3N2 virus antigen.

In a sixth aspect, the invention provides the use of an antibody or antigen-binding fragment of the second or third aspect for the preparation of a medicament for the prevention or treatment of influenza H3N2 virus infection.

In a seventh aspect, the invention provides a specific epitope of hemagglutinin of an influenza virus of subtype H3, which has the amino acid sequence GKNCTLIDALLGDPQ.

In an eighth aspect, the invention provides an isolated antibody or antigen-binding fragment that specifically binds to an epitope described in the seventh aspect, wherein the antibody is preferably an IgG antibody.

In a ninth aspect, the invention provides the antibody or antigen-binding fragment of the eighth aspect, which is a monoclonal antibody or antigen-binding fragment thereof.

In a tenth aspect, the invention provides a method of preparing an immune serum comprising the antibody or antigen-binding fragment of the eighth aspect, the method comprising immunizing an animal with a synthetic conjugate of an epitope of the seventh aspect and a carrier, preferably KLH, and collecting blood and isolating serum after immunization.

In an eleventh aspect, the invention provides use of the antibody or antigen-binding fragment of the eighth or ninth aspect in the preparation of a reagent for detecting an influenza H3N2 virus antigen.

In a twelfth aspect, the invention provides the use of an antibody or antigen-binding fragment of the eighth or ninth aspect for the preparation of a medicament for the prevention or treatment of influenza H3N2 virus infection.

In a thirteenth aspect, the invention provides a polypeptide having the sequence ICDSPHQILDGKNCTLIDALLGDPQ.

In a fourteenth aspect, the present invention provides use of the polypeptide of the thirteenth aspect for the preparation of a reagent for detecting H3N2 influenza virus hemagglutination-inhibiting antibody.

In a fifteenth aspect, the present invention provides use of the epitope of the first or seventh aspect for preparing a reagent for detecting an H3N2 influenza virus hemagglutination-inhibiting antibody.

Drawings

FIG. 1 is a synthetic peptide screening chart for the immunodominant epitope of influenza strain Victoria/210/2009x Puerto Rico/8/1934(NCBI accession number CY 121727).

FIGS. 2A-2C show the reactivity of synthetic peptides P6, P7, P21 with HA antiserum to influenza virus subtypes H1-H16, in that order.

In FIG. 3, A shows the titer of the synthetic peptide antiserum; b shows the reaction of the synthetic peptide antiserum with the purified H1-H16HA 1protein as determined by the Western blot method; c shows a Western blot method to determine the reaction of synthetic peptide antisera with H3-HA (68HA, 77HA, 89HA, 99HA and 07HA) proteins and influenza B virus (IFVB) HA proteins in different years; d shows the ELISA method for determining the reaction of the synthetic peptide antiserum with the purified H1-H16HA 1 protein.

Fig. 4 shows the three-dimensional structural positions and conservation analysis of synthetic peptides P6 and P7.

FIG. 5 shows an immunofluorescence assay for the detection of influenza virus subtype H3 using the P6 and P7 antisera. Wherein a shows the reactivity of P6 antisera with H3N2 infected MDCK cells; b shows the reactivity of P7 antisera with H3N2 infected MDCK cells; c shows the reactivity of the negative mouse sera with H3N 2-infected MDCK cells (negative control); d shows the reactivity of NP antisera with H3N2 infected MDCK cells (positive control).

Detailed Description

The invention will be further illustrated with reference to preferred embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Specific experimental procedures are not noted in the following examples, and are generally performed according to conventional conditions and methods, such as those described in molecular cloning laboratories handbook (Sambrook, et al. New York: Cold Spring Harbor Laboratory Press,1989) or those provided by reagent manufacturers.

Example 1 identification of immunodominant epitopes of H3N2 hemagglutinin protein

1.1 Synthesis of A/H3N2 influenza hemagglutinin protein extracellular region peptide library

51 recombinant H3N2 influenza virus A/reassitant/NYMC X-187(Victoria/210/2009X Puerto Rico/8/1934, NCBI accession number CY121727) strains of hemagglutinin protein extracellular region peptide library were entrusted to Shanghai bioengineering technology Limited. Each peptide is 15 amino acids in length, with each adjacent two peptides overlapping by 5 amino acids. All synthetic peptides were purified by high performance liquid chromatography with > 95% purity and identified by mass spectrometry.

The amino acid positions and sequences of all synthetic peptides are shown in Table 1, since the amino acids 1-16 of the H3N2HA protein are signal peptides, the amino acid positions of the peptides in Table I are calculated from the 17 th amino acid and are denoted as the 1 st amino acid according to the labeling method of the amino acid positions of H3N2 in the literature.

TABLE 1A/H3N2 peptide library of the extracellular region of hemagglutinin protein of influenza virus

1.2 Indirect ELISA screening of immunodominant epitopes

The resulting 51 polypeptides were diluted with a coating solution (pH9.6 carbonate buffer), coated at 1. mu.g/well on a 96-well microplate (Costar Co., Ltd., cat # 9018), and left overnight at 4 ℃. The next day, blocking solution (1% BSA/PBS) was added, the mixture was blocked at 37 ℃ for 2 hours, and then washed 1 time with PBST (PBS containing 0.1% Tween 20) buffer, and the washing solution was dried. Mu.l of 1:1000 diluted anti-H3 HA mouse immune serum was added to each well, incubated at 37 ℃ for 1 hour, drained off and washed 5 times with PBST buffer. Then, 100. mu.l of horseradish peroxidase goat anti-mouse IgG (product of Sigma; cat. No.: A0168) diluted at 1:40000 was added to each well, incubated at 37 ℃ for 1 hour, drained off the liquid, and washed 5 times with PBST buffer. Mu.l of 3,3 ', 5, 5' -Tetramethylbenzidine (TMB) substrate was added to each well and developed in the dark for 15 minutes, and 50. mu.l of 2M H was added₂SO₄The reaction was terminated and the absorbance at 450nm was measured. The results show that the three peptides P6, P7 and P21 can be specifically combined with anti-H3 HA mouse immune serum (figure 1).

1.3 reactivity of dominant epitope peptides with different subtype HA antisera

The three polypeptides P6, P7 and P21 were diluted with a coating solution, coated on a 96-well microplate (Costar; cat # 9018) at 1. mu.g/well, and left overnight at 4 ℃. The next day, blocking solution was added, incubated at 37 ℃ for 2 hours, washed 1 time with PBST buffer, and the wash solution was spun off. Mu.l of 1:1000 diluted anti-H1-H16 HA mouse immune serum was added to each well, incubated at 37 ℃ for 1 hour, drained off the liquid, and washed 5 times with 0.1% PBST buffer. Then each holeMu.l of horseradish peroxidase goat anti-mouse IgG (product of Sigma; cat # A0168) diluted at 1:40,000 was added thereto, incubated at 37 ℃ for 1 hour, then the liquid was discarded, and washed 5 times with PBST buffer. Mu.l TMB substrate per well was developed in the dark for 15 minutes and 50. mu.l 2M H was added₂SO₄The reaction was terminated and the absorbance at 450nm was measured. The results show that the three peptides P6, P7 and P21 can only be specifically recognized by the immune serum of H3HA mice (figure 2), but do not react with other subtype HA antiserum, and the three epitopes are subtype H3 specific epitopes.

Example 2 antigenicity of immunodominant peptides

2.1 conjugation of peptides to KLH

According to the epitope screening result, polypeptides P6, P7, P21 and P16(P16 is a negative reaction control) are synthesized, and are respectively coupled with KLH after a cysteine is added at the C terminal, so as to be used for immunizing mice. Peptide synthesis and coupling to KLH were performed by shanghai biotechnology limited.

TABLE 2 amino acid sequence of peptide-KLH conjugates for immunization

2.2 immunization protocol

6-8 week old female Balb/c mice were immunized with the KLH-conjugated polypeptide described above. The immunization protocol was as follows: a total of 3 immunizations were performed, each at 14-day intervals. For the first immunization, an equal volume of peptide-KLH conjugate was mixed with Freund's complete adjuvant and 100. mu.g of peptide-KLH conjugate was injected subcutaneously per mouse. For boosting, equal volumes of peptide-KLH conjugate were mixed with Freund's incomplete adjuvant and 50. mu.g of peptide-KLH conjugate was injected subcutaneously per mouse. Blood is collected from eyeball 14 days after last immunization, and separated serum is stored at-20 ℃ for later use.

2.3 immune serum Titer assay

Diluting the polypeptides P6, P7, P21 and P16 with coating solution (final concentration of 1 μ g/well), coating enzyme label plate (Costar product, cat # 9018), standing at 4 deg.C overnight, adding blocking solution, incubating at 37 deg.C for 2 hrThe column was washed with PBST buffer 1 time and the wash solution was spun off. Mu.l of the corresponding mouse immune serum serially diluted (from 1:5,000 to 1:320,000) was added to each well, while the non-immune mouse serum was set as a control, incubated at 37 ℃ for 1 hour, drained off the liquid, and washed 5 times with PBST buffer. Then, 100. mu.l of goat anti-mouse IgG horseradish peroxidase (product of Sigma; cat. No.: A0168) diluted 1:40000 was added to each well, incubated at 37 ℃ for 1 hour, the liquid was discarded, and washed 5 times with PBST buffer. Mu.l TMB substrate per well was developed in the dark for 15 minutes and 50. mu.l 2M H was added₂SO₄The reaction was terminated and the absorbance at 450nm was measured. The results are shown in FIG. 3A, which shows that P6, P7 and P16 all induced stronger antibodies with the antibody titers of 1:80,000, 1:40,000 and 1:40,000 as cut-off values 2.1 times the OD450nm of the mouse before immunization, suggesting that P6 and P7 have stronger immunogenicity. However, the P21 polypeptide did not induce significant antibody titers.

Example 3 specificity of epitopes

3.1 expression of H1-H16 subtype HA 1protein and H3HA protein in different years

Because the epitopes P6 and P7 are located in the hemagglutinin HA1 subunit, we used a recombinant baculovirus expression system to express and purify the H1-H16 subtype HA 1protein, respectively, according to literature reported methods (see Cui S, et al, secretion expression of all 16subtypes of the hemagglutinin 1protein of influenza A viruses in the infection cells. J Virol methods.2011,177: 160-7). Meanwhile, the HA extracellular region of H3N2 subtype influenza virus (abbreviated as 68HA, 77HA, 89HA, 99HA and 07HA, respectively) and the HA extracellular region of influenza b virus (IFVB) were expressed and purified using recombinant baculovirus expression systems, respectively, in different years (1968, 1977, 1989, 1999 and 2007).

TABLE 3 hemagglutinin Gene information of influenza virus subtype H3, influenza B virus and influenza virus subtypes at different years

3.2 specificity of epitope immune sera reacting with H3HA protein

3.2.1 specificity of immunoblotting detection epitopes

100ng of purified H1-H16HA 1protein, H3HA protein from different years (68HA, 77HA, 89HA, 99HA and 07HA) and IFVB HA protein were subjected to 12% SDS-PAGE, and then transferred to a nitrocellulose membrane (NC membrane, product of Pall corporation, cat # S80209). NC membranes were blocked with 5% skim milk for 2 hours at room temperature and incubated for 2h with 1:500 dilutions of P6, P7 and P16 immune mouse serum. NC membranes were washed 5 times with PBST and 1:10,000 diluted

Fluor 800-labeled goat anti-mouse IgG secondary antibody (product of Li-Cor, cat. No. 926-32210). After washing the NC membrane thoroughly with PBST, the membrane was washed with

A near infrared imaging system (Li-Cor) was used to scan the membrane and analyzed using the Odyssey software. The results are shown in FIGS. 3B, 3C, P6 and P7 sera from immunized mice specifically reacted only with the H3HA 1protein and not with the hemagglutinin proteins of other subtypes of influenza virus (FIG. 3B). Moreover, P6 specifically bound to 77HA, 89HA, 99HA and 07HA, but did not react with 68 HA; the P7 antiserum specifically bound to all year-derived H3 subtype HA proteins (fig. 3C).

3.2.2 ELISA method for detecting epitope specificity

H1-H16HA 1 was diluted with the coating solution (final concentration: 50 ng/well), coated with an enzyme-labeled plate (product of Costar Co., cat # 9018), incubated at 4 ℃ overnight, added with a blocking solution, incubated at 37 ℃ for 2 hours, washed 1 time with PBST buffer, and the washing solution was dried. Mu.l of serial 1:500 diluted P6, P7 and P16 mouse immune sera were added to each well, incubated at 37 ℃ for 1 hour, spun off and washed 5 times with PBST buffer. Then, 100. mu.l of horseradish peroxidase goat anti-mouse IgG (product of Sigma; cat. No.: A0168) diluted at 1:40,000 was added to each well, incubated at 37 ℃ for 1 hour, drained off the liquid, and washed 5 times with PBST buffer. Mu.l TMB substrate per well was developed in the dark for 15 minutes and 50. mu.l 2M H was added₂SO₄The reaction was terminated and the absorbance at 450nm was measured. The results showed that the P6 and P7 antisera reacted specifically only with H3HA 1protein, but not with other subtypes of influenza virus HA 1protein (fig. 3D).

The above results indicate that P6 is a specific epitope common to influenza viruses of the H3 subtype spanning 30 years, whereas P7 is a specific epitope common to influenza viruses of the H3 subtype spanning 40 years.

Example 4 homology modeling analysis and sequence alignment

The HA amino acid sequence of the H3N2 influenza strain A/reassiortant/NYMC X-187(Victoria/210/2009X Puerto Rico/8/1934) strain was submitted to the Swiss-model database to search for homologous proteins of known spatial conformation. The three-dimensional structure of H3N2HA was modeled by the Swiss-model auto-homology modeling program. And marking the corresponding three-dimensional structure position of the synthetic peptide with strong reactivity by Rasmol software, and determining that the P6 epitope and the P7 epitope are positioned in the head region. The structure is shown in FIG. 4A.

According to the hemagglutinin gene information of H3 subtype strain and H1, H2, H4-H16 subtype influenza viruses, the amino acid sequences corresponding to the P6 and P7 peptides are found, and the amino acid sequences of P6 and P7 are compared by using the MegAlign program of DNAStar software. The results found that the peptides P6 and P7 were conserved in the sequence of HA of subtype H3 in different years, while their amino acid sequences had low homology with the corresponding sequences of HA of other subtypes of influenza virus (fig. 4B). Notably, the P6 peptide showed mutations at amino acids 53, 54, 62, and 63 in 68HA compared to 77HA, 89HA, 99HA, 07HA, while the P7 peptide showed better conservation in H3HA in different years.

Example 5 construction of H3 subtype influenza Virus antigen and antibody diagnostic methods

Immunofluorescence method for detecting virus antigen

MDCK cells were transferred to a 96-well plate (product of Costar Co., Ltd.; cat # 3599) and cultured in a 5% CO2 incubator at 37 ℃. The next day MDCK cells were infected with H3N2 strain at MOI of 0.1, cultured in a 5% CO2 incubator at 33 ℃ for 48H, H3N2 infected MDCK cells were fixed with 4% paraformaldehyde at room temperature for 30min, and mouse anti-P6 and mouse anti-P7 peptide antibodies were added, incubated at 37 ℃ for 1H, washed with PBST, added with the corresponding fluorescent secondary antibody, incubated at 37 ℃ for 1H, washed with PBST, and then subjected to scanning analysis using an Operetta high content screening system. The results showed that mouse anti-P6 peptide, mouse anti-P7 peptide antibody, and influenza NP antibody (as positive controls) all specifically recognized H3N 2-infected MDCK cells, while negative mouse control sera failed to recognize H3N 2-infected MDCK cells (fig. 5).

Because the peptides P6 and P7 have 5 amino acid overlaps, we synthesized a polypeptide with amino acid sequence at position 51-75 (named P6P7), which was prepared by coating the peptides P6, P7 and P6P7 on enzyme-linked plate, incubating overnight at 4 deg.C, adding blocking solution, incubating at 37 deg.C for 2 hours, washing 1 time with PBST buffer, and spin-drying the washing solution. Mu.l of serial 1:400 dilution of H3N2 Hemagglutination Inhibition (HI) antibody positive healthy human serum was added to each well, incubated at 37 ℃ for 1 hour, drained off the liquid, and washed 5 times with PBST buffer. Then, 100. mu.l of horseradish peroxidase goat anti-human IgG (product of Sigma; cat. No. A8792) diluted at 1:40,000 was added to each well, incubated at 37 ℃ for 1 hour, centrifuged off, and washed 5 times with PBST buffer. Mu.l TMB substrate per well was developed in the dark for 15 minutes and 50. mu.l 2M H was added₂SO₄The reaction was terminated and the absorbance at 450nm was measured. The results showed that in 92H 3N2 HI antibody positive healthy human sera, 79 were positive for P6 peptide IgG antibody, 80 were positive for P7 peptide IgG antibody, and 82 were positive for P6P7 peptide IgG antibody, with coincidence rates of 85.9%, 87%, and 89.1%, respectively (table 4).

TABLE 4 comparison of the peptide-ELISA test method with the hemagglutination inhibition test method

a represents the number of positives and b represents positivity.

Claims

1. A specific epitope peptide of hemagglutinin of H3 subtype influenza virus has the amino acid sequence ICDSPHQILDGKNCT or GKNCTLIDALLGDPQ.

2. A polypeptide having the sequence ICDSPHQILDGKNCTLIDALLGDPQ.

3. Use of the polypeptide of claim 2 for preparing a reagent for detecting a hemagglutination-inhibiting antibody to an influenza H3N2 virus.

4. Use of the epitope peptide of claim 1 for producing a reagent for detecting a hemagglutination-inhibiting antibody against an influenza H3N2 virus.