CN109232721B

CN109232721B - Peptide segment 418-429 of highly conserved region of HCV envelope protein and application thereof

Info

Publication number: CN109232721B
Application number: CN201811121832.3A
Authority: CN
Inventors: 邓凯
Original assignee: Guangzhou 8th People's Hospital
Current assignee: Guangzhou 8th People's Hospital
Priority date: 2018-09-26
Filing date: 2018-09-26
Publication date: 2021-06-29
Anticipated expiration: 2038-09-26
Also published as: CN109232721A

Abstract

The invention belongs to the technical field of biomedical engineering, and particularly relates to a peptide 418-429 of a highly conserved region of HCV envelope protein and application thereof. The peptide segment of the highly conserved region of the HCV envelope proteins provided by the invention is the peptide segment 418-429 of the highly conserved region of 1 HCV envelope proteins, which is provided by comparing the highly conserved region of the HCV envelope proteins through a sequence and combining the spatial structure of the envelope proteins, and the amino acid sequence is Gly-Ser-Trp-His-Ile-Asn-Ser-Thr-Ala-Leu-Asn-Cys, and animal experiments prove that the peptide segment can induce a high-efficiency antibody with broad-spectrum neutralization effect on 1-6 genotype HCV in vivo. The highly conserved peptide segment 418-429 derived from the HCV envelope protein can also be used for preparing vaccines for preventing and treating hepatitis C, and has the advantages of high conservation, wide application range and the like.

Description

Peptide segment 418-429 of highly conserved region of HCV envelope protein and application thereof

Technical Field

The invention belongs to the technical field of biomedical engineering, and particularly relates to a peptide 418-429 in a highly conserved region of HCV envelope protein and application thereof.

Background

Hepatitis C Virus (HCV) belongs to the family flaviviridae single-stranded positive-strand RNA virus. HCV infects about 3% of the world's population, with 75-85% of these developing chronic hepatitis and being at risk for liver cirrhosis and cancer. In addition, HCV has very high genetic variability and can be classified into 1-7 genotypes depending on its genomic phylogenetic and sequence differences. The dominant genotypes are not the same in different countries and regions, and 1b and 2a genotypes are common in China. Due to the successful development of Direct acting antiviral Drugs (DAAs), the treatment of hepatitis c has achieved good results. However, there is still a lack of vaccines for preventing HCV infection. According to the estimation of hepatitis C prevention and treatment guidelines (2015 updated edition) in China, about 1000 ten thousand patients with hepatitis C exist in China. Since a significant proportion of patients cannot pay the high medical costs of DAA and cannot be completely cured, they are a potential source of HCV infection. Even if patients are completely cured by DAA, there is still a risk of re-infection by HCV due to the high variability of the HCV genes. Therefore, the development of an effective anti-HCV vaccine is both an important scientific problem and an urgent social public health problem.

During HCV infection, it is introduced into the cell via the interaction of envelope proteins with protein molecules such as hepatocyte receptors SRB1, CD81, Claudin-1 and Occludin. Meanwhile, the HCV envelope protein is also a main target for stimulating host humoral immunity and generating protective neutralizing antibodies. However, the effectiveness of using HCV envelope proteins as vaccine immunogens is not optimal because of the high variability of the HCV envelope proteins, which in fact does not ensure protection against HCV of any genotype; meanwhile, a highly variable region such as highly variable region 1 (HVR 1) is present in the envelope protein, and studies have shown that this region cannot induce the production of broadly neutralizing antibodies and interferes with the protective action of neutralizing antibodies (see: Keck Z. Y., et al. doi:10.1128/JVI V II.02.2016, 90; PrentoeJ, Velazquez-Moctezuma R, Foung S K, Law M & Bukh J. Heaptology 64,2016, 1881-1892). Vaccines that use the core, NS3, NS4, and NS5 proteins of HCV as immunogens to elicit cellular immunity are also under development and their efficacy is yet to be verified (see Houghton M. immunological reviews 239,2011, 99-108). A clinical trial at stage 1/2 was also being conducted to investigate vaccines that simultaneously express HCV structural and non-structural proteins using DNA vectors (see the literature: Liang T.J. Nature media 19,2013, 869-878). To date, there is no data showing that the above vaccines can completely protect a host from infection by HCV. The development of vaccines against HCV is of great importance.

In addition to using the entire envelope protein as a vaccine immunogen, another approach is to use the peptide fragment it contains for immunoassay. Torresi et al found that antiserum generated by immunizing mice with certain envelope protein peptide fragments could effectively block the H77 strain pseudovirus model of genotype 1 from binding to hepatocytes (see Torresi J.et al. Immunology and cell biology 85,2007,169-173), suggesting that peptide fragment immunization could also be used as a vaccine development concept (see El-Awady M.K.et al. Virology J.6, 2009, 66). Although the HCV envelope proteins are highly variable, they still contain some highly conserved regions. We have previously performed an analysis using 536 sequences covering 1-6 genotypes of HCV and found several highly conserved sites or regions of the HCV envelope protein (see literature: Deng K.et al. The peptide segment of the site or the region with high conservation of the envelope protein is used as immunogen and can be used as a novel method for developing HCV vaccine.

In previous studies, conservative evaluation of 1a, 1B, 4d genotype HCVE2 sequences in NCBI protein databases and B-cell epitope prediction have been performed to synthesize epitope polypeptides, and several HCV genotype-specific epitope polypeptides were screened, suggesting that they may be candidate immunogens for preventing HCV infection (Qilingxia, Jilin university, 2015). There have also been reports of using computer software to predict the effective epitope of HCV envelope proteins (Zhengyu, Suqin, Linfang, etc., 2002, 22(2): 202-. However, these studies have not verified whether the epitope peptides can induce high titers of antibodies in vivo or whether the antibodies have a preventive effect on HCV infection of 1-6 genotypes in an infection model.

Chinese patent CN103113458B discloses a hepatitis C virus B cell epitope peptide, which discloses a peptide segment PUH134 and application thereof, the position of the epitope peptide is 519-533, the epitope peptide provides a new solution for the development of HCV therapeutic antibodies and the development of HCV preventive vaccines, but the epitope peptide only aims at 1B gene subtype, the action range is small, and the conservation of the epitope peptide is poor due to the high variability of HCV envelope protein.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a peptide fragment 418-429 derived from a highly conserved region of HCV envelope protein and application thereof. The peptide segment derived from the highly conserved region of the HCV envelope protein provided by the invention has high conservation, can induce the generation of broad-spectrum neutralizing antibody, and provides a new antigen design for researching and developing hepatitis C vaccines.

In order to achieve the aim, the invention provides a peptide segment 418-429 of a highly conserved region of HCV envelope protein, the amino acid sequence of which is Gly-Ser-Trp-His-Ile-Asn-Ser-Thr-Ala-Leu-Asn-Cys.

The invention also provides a drug composition for resisting 1-6 type HCV, wherein the 1-6 type HCV drug composition comprises a peptide segment 418-429 of a highly conserved region on the HCV envelope protein and a pharmaceutically acceptable medicinal carrier; the medicinal carrier is water for injection.

The 1-6 type HCV medicine composition provided by the invention also comprises pharmaceutic adjuvants and pharmaceutically acceptable salts; the pharmaceutic adjuvant comprises a lubricant, namely simethicone, a cross-linking agent, namely sodium alginate, an adhesive, namely ethyl cellulose, and a preservative, namely ethylparaben; the pharmaceutically acceptable salt is barbiturate.

The preparation method of the 1-6 type HCV medicine composition comprises the following steps:

a. 536 sequences covering 1-6 genotypes of HCV (see the literature: Deng K.et al.. PloS one 10,2015, e0138756) are obtained from NCBI website and are compared to obtain a peptide segment 418-429 of a highly conserved region on HCV envelope protein, and the peptide segment is synthesized by Shanghai Gill biochemical company to obtain an amino acid peptide segment; the purity of the amino acid peptide fragment is 99 percent;

b. heating 600mL of water for injection to 70 ℃, adding 0.1g of the amino acid peptide segment obtained in the step a, and centrifuging at 300r/min for 10min until complete dissolution to obtain a mixture I;

c. b, adding 10g of simethicone, 1.5g of sodium alginate, 0.8g of ethyl cellulose and 1.5g of ethylparaben into the mixture I obtained in the step b, maintaining the temperature at 70 ℃, and centrifuging for 20min at 500r/min until the mixture is completely dissolved to obtain a mixture II;

d. adding 8g of barbiturate into the mixture II, stirring at 500r/min until the barbiturate is completely dissolved, and cooling to 30 ℃ to obtain the barbiturate-containing solid preparation.

The invention also provides the application of the vaccine for preventing and treating hepatitis C, which is prepared by coupling the peptide segment 418-429 of the highly conserved region of the envelope protein with KLH (Klenow-Loeve H) as an immune antigen, assisting with a Freund's adjuvant, and adding a preservative of thimerosal, an inactivator of formaldehyde, a stabilizer of lactose, phosphate buffer salt and the like.

The preparation method of the preventive and therapeutic hepatitis C vaccine comprises the following steps:

firstly, coupling a peptide segment 418-429 in a highly conserved region of HCV envelope protein with KLH to obtain a coupling protein; the coupling process of the peptide fragment and KLH is carried out according to the manufacturer's instructions of KLH-peptide coupling kit of CellMosaic;

introducing the coupling protein obtained in the step one into an isolated chimpanzee hepatocyte to obtain a recombinant chimpanzee hepatocyte;

adding an inactivating agent formaldehyde into the recombinant chimpanzee liver cells obtained in the step (ii), and treating for 30 hours at 37 ℃ to obtain inactivated recombinant chimpanzee cells;

fourthly, adding a Freund type complete adjuvant into the inactivated recombinant chimpanzee liver cells obtained in the third step, fully mixing and subpackaging to obtain an inactivated vaccine;

fifthly, adding thimerosal into the inactivated vaccine obtained in the step IV according to the weight ratio of the thimerosal: lactose: the mass ratio of the phosphate buffer salt is 1: 3: 2, fully mixing, subpackaging, freezing and drying in vacuum to prepare the dry vaccine.

The invention also provides a method for inducing B cells to generate antibodies with broad-spectrum neutralization resistance to the 1-6 type HCV by using the peptide segment 418-429 with the highly conserved region and KLH coupled as an immunizing antigen.

The method for obtaining the neutralizing resistant antibody comprises the following steps:

s1, mixing the immune antigen and adjuvant in equal volume to obtain immunogen;

s2, immunizing Balb/c mice of 8-10 weeks old by the intraperitoneal subcutaneous multi-point injection method for the immunogens obtained in the step S1 respectively at the 0 th week, the 2 th week, the 4 th week, the 5 th week, the 6 th week, the 7 th week and the 8 th week for 7 times, adding 125 mu L of Freund 'S complete adjuvant at the 1 st time, adding 62.5 mu L of Freund' S incomplete adjuvant at the later 6 times, adding 250 mu L of emulsified antigen at the 1 st time of each mouse, wherein the antigen amount is 250g, adding 125 mu L of emulsified antigen at the later 6 times, and the antigen amount is 125 g;

and (4) after S3 immunization is finished, collecting the antibody in the serum to obtain the antibody.

The invention also provides a blocking agent, wherein the blocking agent is an antibody aiming at the peptide segment 418-429 in the highly conserved region, and can efficiently block the invasion of the HCV virus to normal cells.

Compared with the prior art, the highly conserved peptide segment 418-429 of the HCV envelope protein provided by the invention has the following advantages:

(1) the peptide segments of the highly conserved region of the HCV envelope protein provided by the invention are highly conserved in 1-6 HCV genotypes;

(2) the peptide segment of the highly conserved region of the HCV envelope protein can be used for preparing a preventive and therapeutic hepatitis C vaccine, and provides a new antigen design for researching and developing the hepatitis C vaccine;

(3) the peptide fragment of the highly conserved region of the HCV envelope protein provided by the invention can be used as vaccine immunogen, can induce the generation of broad-spectrum neutralizing antibody, and can effectively prevent the infection of 1-6 genotype HCV cell infection model (HCVcc).

Drawings

FIG. 1 shows the result of ELISA detection of the binding of the peptide fragment to serum collected at 9 weeks of the mice immunized with 418-429/KLH peptide fragment;

FIG. 2 shows the result of ELISA detection of the binding of the peptides to the serum collected at week 9 from the mice immunized with peptides 685-693/KLH;

FIG. 3 shows the neutralizing effect of antiserum from mice immunized with peptide 418-429 on infection with strain J6/JFH1 (genotype 2a) of HCV at different dilutions;

FIG. 4 shows the effect of sera from mice immunized with peptide 418-429 on the neutralizing activity and IC50 value of the HCV genotype 1 TNcc strain at different dilutions;

FIG. 5 shows the effect of the sera of mice immunized with peptide 418-429 on the neutralizing activity and IC50 value of the strain of HCV gene type 2J 6/JFH1 at different dilutions;

FIG. 6 shows the effect of sera from mice immunized with peptide 418-429 on the neutralizing activity and IC50 value of the HCV genotype 3 strain S52 at different dilutions;

FIG. 7 shows the effect of sera from mice immunized with peptide 418-429 on the neutralizing activity and IC50 value of the strain ED43 of HCV gene type 4 at different dilutions;

FIG. 8 shows the effect of sera from mice immunized with peptide 418-429 on the neutralizing activity and IC50 value of the SA13 strain of HCV genotype 5 at different dilutions;

FIG. 9 shows the effect of sera from mice immunized with peptide 418-429 on the neutralizing activity and IC50 value of HK6a strain, HCV gene type 6, at different dilutions.

Detailed Description

The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

Example 1A pharmaceutical composition comprising the highly conserved domain peptide 418-429 of HCV

The drug composition containing the HCV highly conserved domain peptide segment 418-429 comprises the highly conserved domain peptide segment 418-429 on an HCV envelope protein, a drug carrier is water for injection, a drug adjuvant lubricant simethicone, a cross-linking agent sodium alginate, an adhesive ethylcellulose, a preservative ethylparaben and a pharmaceutically acceptable barbiturate.

a. 536 sequences covering 1-6 genotypes of HCV (see the literature: Deng K.et al.. PloS one 10,2015, e0138756) are obtained from NCBI website and are compared to obtain a peptide segment 418-429 of a highly conserved region on HCV envelope protein, and the peptide segment is synthesized by Shanghai Gill biochemical company to obtain an amino acid peptide segment;

Example 2A vaccine for the prevention and treatment of hepatitis C

The vaccine for preventing and treating hepatitis C comprises a coupling protein obtained by coupling a peptide segment 418-429 in a highly conserved region of the envelope protein with KLH, a Freund type adjuvant, a preservative of thimerosal, an inactivator of formaldehyde, a stabilizer of lactose and phosphate buffer salt.

EXAMPLE 3 Balb/c mice immunization

Downloading 1-6 genotype HCV sequences with 536 strips through NCBI (https:// www.ncbi.nlm.nih.gov /) website, and obtaining a peptide segment 418-429 of the highly conserved region of HCV envelope protein by performing sequence analysis on the website https:// www.ebi.ac.uk/Tools/msa/clustalo/; meanwhile, 685-693 is located in the transmembrane region of the envelope protein and is not exposed outside, and theoretically, a neutralizing antibody cannot be generated, so that the peptide fragment is used as a negative control peptide fragment in the experiment of the invention.

The peptide segment 418-429 and the negative control peptide segment 685-693 are synthesized by Shanghai Jier Biochemical company, the purity is up to more than 99%, and the peptide segment is coupled with KLH to be used as an immune antigen.

2 peptide fragment immune antigens respectively immunize 6 Balb/c mice with age of 8-10 weeks, 6 non-immunized negative control mice are arranged, the specific immune process and the dosage of the antigens and the adjuvants are shown in table 1, the abdominal cavity or subcutaneous multi-point injection is carried out, a commercial Freund's complete adjuvant needs to be oscillated and shaken before use, so that the precipitated mycobacteria are fully and uniformly mixed, the antigens and the adjuvant are required to be mixed together in equal volume, the mixture is ground into a water-in-oil chyle mixture, and a drop is placed on the water surface and is not easy to immediately diffuse into a droplet shape, which indicates that the water-in-oil state is achieved.

During immunization, the antibody production titers were monitored by orbital bleeding ELISA. At week 9, mice were sacrificed and serum was diluted at fold ratio to detect antibody titers after blood collection; and purified antibody IgG for detection of its neutralizing activity against HCVcc genotype 1-6.

TABLE 1 Balb/c mice immunization 3 polypeptide antigens

Example 4 ELISA detection of mouse serum antibodies

The ELISA detection of the mouse serum antibody comprises the following specific operation steps:

(1) wrapping a plate: streptavidin was dissolved in carbonate-bicarbonate buffer at pH 9.6 to a final concentration of 10. mu.g/mL and the streptavidin solution was coated onto 96-well ELISA plates at 100. mu.L/well overnight at 4 ℃. The next day, the plate was washed three times with phosphate balanced physiological saline (PBS), 300. mu.L/well, 5 minutes/time;

(2) and (3) sealing: blocking at 37 ℃ for 1 hour by 200 mu L of blocking solution (goat serum-PBS with the volume fraction of 10%) per well, washing the plate with PBS for three times, 300 mu L per well and 5 minutes per time;

(3) peptide fragment binding: adding 100 mu L of 10 mu g/mL biotin-labeled peptide fragment into a 96-well plate, incubating for 2 hours at 37 ℃, washing the plate with PBS for three times, 300 mu L/well and 5 minutes/time;

(4) binding of the test antibody: adding 100 μ L of mouse serum with different dilutions (PBSTG dilution: PBS containing 0.05% Tween 20 and 10% goat serum) into a 96-well plate, incubating at 37 ℃ for 2 hours, wherein each serum is provided with 3 parallel wells, and a normal mouse control serum and a blank group without serum are simultaneously arranged, wherein PBST (PBS containing 0.05% Tween 20) washes the plate 4 times, 300 μ L/well, 5 minutes/time;

(5) and (3) binding of a secondary antibody: mu.L/well horseradish peroxidase-labeled goat anti-mouse IgG antibody (1/5000, PBSTG dilution) was added to the 96-well plate, incubated at 37 ℃ for 1 hour, and the plate was washed 4 times with PBST, 300. mu.L/well, 5 minutes/time.

(6) Color development: the o-phenylenediamine hydrochloride substrate was dissolved in citrate buffer (0.05M) to a final concentration of 0.4. mu.g/mL, and 40. mu.L of 3% H was added₂O₂Mixing evenly, adding into 96-well plate, reacting at room temperature for 30 min at 100 μ L/well, and terminating reaction at 100 μ L/well with 2N HCl;

(7) reading: after the reaction was terminated, the OD was read on an ELISA plate reader of Tecan, Switzerland and the reading was recorded.

The results show that: peptide 418-429 and negative control peptide 685-693 both successfully induced the production of specific antibodies. The OD values of ELISA assays for serum-bound peptidyl antigen taken at week 9 of immunization of mice are shown in FIGS. 1-2.

Example 5 purification of mouse serum antibody IgG

To confirm that the immunization with the peptide 418-429 indeed generates antibodies with neutralizing activity, and to exclude the influence of other factors in serum on the experimental results. We purified serum IgG and performed a neutralization assay. Mouse serum was purified using Magne from Promega, USA^TMProtein G Beads for Antibody Purification kit (Cat. G7471), the procedure was performed according to the manufacturer's instructions.

Example 6 culture of HCV cell infection model (HCVcc)

HCVcc strains and genotypes used in this experiment included TNcc (1a), J6/JFH1(2a), S52(3a), ED43(4a), SA13(5a) and HK6a (6 a). The culture and collection method comprises the following steps:

(1) plate preparation: huh7.5 cells were seeded into 25T cell culture flasks at 2X 10⁶Putting each cell/bottle into a cell culture box, and infecting when the cell density is about 50% the next day;

(2) infection: the culture supernatants of full length HCV RNA transfected Huh7.5 cells from each genotype were added to 25T cell culture flasks at a multiplicity of infection of 0.01, and the infected cells were passaged every 3 days at a ratio of 1: 3;

(3) collecting viruses: 14 days after infection, cell culture supernatants were collected, centrifuged at 4000 rpm for 5 minutes at 4 ℃ and filtered through a 0.45 μm filter, and the filtrate was collected and stored at-80 ℃.

Example 7 neutralizing Activity of mouse serum IgG on different genotypes of HCVcc

(1) Plate preparation: huh7.5 cells were seeded into 96-well plates at a density of 6X 10³Putting the cell culture box into a hole, and infecting the cell culture box the next day;

(2) infection: HCVcc of different genotypes of 100FFU was mixed with mouse IgG at different concentration gradients (2-fold dilution from 100. mu.g/mL to 0.05. mu.g/mL) and incubated for 1 hour at 37 ℃; after incubation, the mixed solution is added into the holes of a 96-well plate, 100 mu L/hole, and each dilution concentration is made into 2 parallel holes; after incubation for 6 hours at 37 ℃, the mixed solution is completely sucked and replaced by a fresh complete culture medium, and incubation is carried out for 48 hours at 37 ℃;

(3) formaldehyde fixation: the medium in the wells was aspirated off, washed 1 time with PBS, 300. mu.L/well, and fixed with 4% paraformaldehyde at 4 ℃ for 15 minutes;

(4) cell perforation: adding 0.5% Triton X-100-PBS buffer solution into a plate hole, incubating for 20 minutes at room temperature, and washing the plate for 3 times by 0.05% Tween 20-PBS for 5 minutes each time;

(5) and (3) sealing: 5% BSA-0.05% Tween 20-PBS blocking solution was added to the wells and incubated for 2 hours at room temperature. Washing the plate with 0.05% Tween 20-PBS for 3 times, 5 minutes each time;

(6) primary antibody binding: 1:150 primary antibody (diluted by 5% BSA-0.05% Tween 20-PBS blocking solution) is added into a plate hole, incubated for 2 hours at room temperature, and washed for 4 times by 0.05% Tween 20-PBS for 5 minutes each time;

(7) and (3) binding of a secondary antibody: 1:200 secondary antibody (diluted with 5% BSA-0.05% Tween 20-PBS blocking solution) is added into a plate hole, incubated for 2 hours at room temperature, and washed for 4 times with 0.05% Tween 20-PBS for 5 minutes each time;

(8) and (3) observation by a fluorescence microscope: the FFU numbers were observed under a fluorescent microscope to calculate the infection rate and IC50 for mouse IgG neutralization.

As a result: the antiserum generated by the peptide fragment 418-429 was able to effectively block the infection of HCV, while the antibody generated by the negative control peptide fragment 685-693 was unable to block the infection (FIG. 3). Further purification of serum IgG induced by mice immunized with the peptide 418-429 showed different degrees of neutralizing activity against 1-6 genotype HCVcc (FIGS. 4-9), indicating that the peptide 418-429 could effectively induce a protective humoral immune response against HCV envelope proteins.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

<110> eighth national hospital in Guangzhou City

<120> peptide segment 418-429 of highly conserved region of HCV envelope protein and application thereof

<130> 2018.9.5

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

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<212> PRT

<213> Hepatitis C Virus (Hepatitis C virus)

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Gly Ser Trp His Ile Asn Ser Thr Ala Leu Asn Cys

1 5 10

Claims

1. A vaccine for resisting HCV type 1-6, which comprises a peptide segment of a highly conserved region of HCV envelope proteins, wherein the amino acid sequence of the peptide segment is Gly-Ser-Trp-His-Ile-Asn-Ser-Thr-Ala-Leu-Asn-Cys.

2. A pharmaceutical composition against HCV type 1-6 comprising the peptide of claim 1 and a pharmaceutically acceptable carrier.

3. Use of a peptide stretch as described in claim 1 for the preparation of a vaccine for the prevention and/or treatment of HCV.

4. The use of claim 3, wherein said peptide fragment induces B cells to produce neutralizing antibodies.

5. A blocking agent against HCV type 1-6, comprising the peptide of claim 1.