CN118184750A - Immunogenic compositions of hepatitis C virus and uses thereof - Google Patents

Abstract

The present invention provides a method of preparing an immunogenic composition comprising treating HCV pseudoparticles with alpha 2-3,6,8,9 neuraminidase a to produce an immunogenic composition. Furthermore, the immunogenic composition is formulated as a vaccine for eliciting an immune response to HCV in a subject.

Description

Immunogenic compositions of hepatitis C virus and uses thereof

Technical Field

The present invention provides an immunogenic composition comprising a Hepatitis C Virus (HCV) pseudoparticle treated with alpha 2-3,6,8,9 neuraminidase A. Furthermore, the present invention provides a use of an immunogenic composition comprising HCV pseudoparticles treated with alpha 2-3,6,8,9 neuraminidase a for the preparation of an HCV vaccine.

Background

Hepatitis C Virus (HCV) is the major causative agent of chronic liver disease, affecting about 1.7 hundred million people worldwide. Most (about 85%) HCV-infected individuals become chronically infected and are at risk of developing cirrhosis and hepatocellular carcinoma. HCV is a member of the flaviviridae family, which is a enveloped virus containing positive-strand genomic RNA. Based on the similarity of genomic sequences, HCV can be divided into six major genotypes and numerous subtypes. The HCV RNA genome comprises an open reading frame located at both the 5 'and 3' ends of the untranslated region (UTR). The positive-strand genomic RNA encodes a polyprotein of about 3010 amino acids, which is co-translated and translated by host and viral proteases into structural proteins (C, E, E2, and p 7) and non-structural proteins (NS 2, NS3, NS4A, NS4B, NS A and NS 5B).

Viral attachment and entry, which represents the first interaction of the virus with the host cell, is the primary goal of adaptive humoral responses. The viral proteins responsible for cell attachment and HCV entry are glycoproteins E1 and E2. Viral proteins are recognized as foreign bodies by the host's immune system and induce antibody production. A small fraction of these antibodies exhibit antiviral activity in vitro and are defined as virus neutralizing antibodies. Neutralizing antibody responses generally provide a first line of adaptive defense against infection by limiting viral transmission. Thus, expression of these glycoproteins has important applications in vaccine discovery and drug targeting. Thus, understanding the genotype and quasimial variation of the viral glycoprotein characteristics (quasispecies variation) is important for understanding the structure-function relationship of proteins.

The envelope glycoproteins E1 and E2 are natural targets for neutralizing antibodies. E2 is a preferred target for humoral and cell-mediated immune responses. Not surprisingly, most of the HCV sequence variation is concentrated in the highly variable region of E2. These regions are known to exhibit a high degree of variability, which can be used to distinguish HCV isolates of the same subtype from quasispecies confirmation (coexistence of different sequences in the same patient).

Glycans (glycans) associated with the viral envelope have a major role in masking neutralizing epitopes (epitopes) and regulating the overall immunogenicity of viral particles. Viral proteins are typically glycosylated by one of three different mechanisms. These processes not only differ among the cellular enzymes involved, but also produce different types of glycan structures. The resulting glycans are referred to as N-linked, O-linked, or glycosyl phosphatidylinositol (glycosylphosphatidylinositol, GPI) anchored. During N-linked glycosylation, the glycan chains are added to the viral protein through asparagine (asparagine) residues. N-linked glycosylation occurs at the site in the protein where the consensus amino acid sequence Asn-X-Ser/Thr is present. This is by far the most common means of glycosylation of viral proteins, which undergo N-linked glycosylation in a process similar to that which occurs on cellular glycoproteins.

Most glycosylation sites on HCV envelope glycoproteins are conserved, and certain glycans associated with these proteins have been shown to play an important role in protein folding and HCV entry. Such highly expressed glycosylation suggests that these glycans can limit the immunogenicity of HCV envelope proteins and limit the binding of certain antibodies to their epitopes on the surface of the virion, as observed in Human Immunodeficiency Virus (HIV) gp 120. According to reports, at least three glycans on E2 (E2N 1, E2N6 and E2N 11) reduce the sensitivity of HCVpp to antigen neutralization, and these glycans also reduce CD81 access to its E2 binding site. In contrast, there is no evidence that the N-linked glycans of E1 help mask the neutralizing epitope. These data suggest that glycans E2N1, E2N6 and E2N11 are able to access the binding site of CD81 and regulate binding of CD81 and neutralizing antibodies to E2. Briefly, this work points out that this region is the primary target for neutralizing antibodies, and HCV glycans help HCV to evade humoral immune responses.

The discovery of anti-HCV compounds and development of HCV vaccines is severely hampered by the lack of cell culture replication systems. The advent of subgenomic replicons that mimic the intracellular response leading to HCV genome replication has enabled the discovery of HCV protease and polymerase inhibitors since the late 1990 s, but failed to study HCV entry or entry inhibitors. Recently, the function of HCV E1E2 in cell attachment and entry (which summarises the whole HCV replication cycle) has been studied using a retroviral-based pseudoparticle (pseudoparticle, pp) assay, in which the infectivity of retroviral particles is conferred by HCV E1E2 envelope proteins. These new experimental systems have made rapid progress in cognition about how HCV glycoproteins E1 and E2 mediate receptor binding and viral entry. These systems facilitate the discovery of a range of viral receptors. This HCV _pp assay is particularly useful in functional assays for HCV E1E2 derived from patients infected with different genotypes and virus subtypes, in profiling the role of the key E2 receptors CD81 and SR-B1 in viral entry, and in measuring the ability of antibodies and patient serum to neutralize infection of cells targeted by HCV _pp.

Recently, infectious pseudoparticles assembled by expressing unmodified HCV envelope glycoproteins on retroviral core particles have been successfully produced. HCV pseudoparticles (HCV pseudoparticles, HCV _pp) were made by transfection of 293T cells with three expression vectors encoding the E1E2 polyprotein, the retroviral core protein, and the packagable retroviral-derived genome with a marker gene, respectively. Surprisingly, infectious pseudoparticles can also be produced without any modification of the HCV envelope glycoprotein. The data accumulated on these pseudo particles strongly suggests that they may mimic the early steps of HCV infection. In fact, they exhibit preferential tropism for hepatocytes (tropism), and they can be neutralized by anti-E2 monoclonal antibodies as well as by serum specificity of HCV-infected patients. Thus, HCV _pp represents the best tool currently available for studying functional HCV envelope glycoproteins and provides a model system for studying HCV cell entry. The development of HCV _pp provides the possibility of HCV neutralization studies with confirmed HCV envelope glycoprotein sequences, and the use of HCV _pp in neutralization studies has been validated. The above description has shown that the progressive appearance of relatively strong neutralization reactions is associated with a reduction in viremia.

Disclosure of Invention

The present invention provides a method of preparing an immunogenic composition comprising treating HCV pseudoparticles with alpha 2-3,6,8,9 neuraminidase a to produce an immunogenic composition. The present invention also provides an immunogenic composition comprising the HCV pseudoparticles treated with alpha 2-3,6,8,9 neuraminidase a. Furthermore, the present invention provides a use of an immunogenic composition comprising HCV pseudoparticles treated with alpha 2-3,6,8,9 neuraminidase a for the preparation of an HCV vaccine.

Viral attachment and entry, representing the first interaction of the virus with the host cell, is the primary goal of adaptive humoral responses. The viral proteins responsible for cell attachment and entry into HCV are glycoproteins E1 and E2. The present invention proposes deglycosylation of E1 and E2 by glycosidases (glycosidase), such as alpha 2-3,6,8,9 neuraminidase A (a broad specificity glycosidase which cleaves linear and branched non-reducing terminal sialic acid (SIALIC ACID) residues from glycoproteins, glycopeptides (glycopeptides) and oligosaccharides (oligosacccharides), which not only increases the sensitivity of detecting neutralizing antibody activity, but also initiates an immune response recognizing higher titres of E1 and E2 variants. To investigate the immunogenicity and protective capacity of HCV pseudoparticles treated with alpha 2-3,6,8,9 neuraminidase a, a mouse model was used. In the present invention, antisera from HCV _Neura-A immunized mice were tested for their ability to inhibit HCV _pp from entering huh7.5 cells (represented by the inhibition of infection rate of huh7.5 cells by HCV _pp). The present invention shows that immunization with HCV _Neura-A can induce an effective systemic humoral response with neutralizing activity compared to the control group, thus effectively inhibiting the infection rate of huh7.5 cells by HCV _pp. The present invention points out that antisera from HCV _Neura-A immunized mice can effectively neutralize HCV _pp, thereby inhibiting HCV _pp (carrying a complete and unmodified envelope glycoprotein of parental origin) from entering cells, and thus inhibiting huh7.5 cell infection. Accordingly, based on the results obtained from the neutralization studies described above, deglycosylation of HCV _pp can be used to design an effective vaccine against HCV infection.

The strategy of the invention (hydrolysis of sialic acid residues of glycoprotein on HCV _pp by alpha 2-3,6,8,9 neuraminidase a) opens up a new direction for vaccine design and can be used with other different vaccine strategies to facilitate development of anti-HCV vaccines.

The terms "a" or "an" are used herein to describe the elements and components of the present invention. This term is used for descriptive convenience only and gives a basic idea of the invention. The description should be read to include one or at least one and the singular also includes the plural unless it is clear that it is meant otherwise. The terms "a" and "an" when used in conjunction with the term "comprising" in the claims may mean one or more.

The term "or" as used in the claims means "and/or" unless explicitly indicated to the contrary, only other options are indicated or unless the other options are mutually exclusive.

The present invention provides a method of preparing an immunogenic composition comprising a viral antigen of Hepatitis C Virus (HCV), comprising: (a) Providing HCV pseudoparticles (HCV pseudoparticles, HCV _pp); (b) Treating the HCV pseudoparticle with α2-3,6,8,9 neuraminidase a to remove sialic acid on the surface of the HCV pseudoparticle; and (c) isolating the HCV pseudoparticles produced in step (b) to produce the immunogenic composition.

In the present invention, the method for preparing HCV pseudo particles comprises: (1) Delivering an expression construct into the cell, wherein the expression construct comprises sequences encoding HCV glycoproteins E1 and E2, a retroviral core protein, and a retroviral derived genome; and (2) isolating HCV pseudoparticles produced by said cells.

In one embodiment, the sequences encoding HCV glycoproteins E1 and E2 comprise the sequences set forth in SEQ ID NO:1, and a sequence shown in 1.

In the method for producing HCV pseudoparticles, three independent expression constructs comprising three nucleic acid sequences encoding retroviral core, genome and hepatitis virus glycoprotein can be designed; alternatively, a construct may be designed which expresses a sequence comprising different nucleic acids. Such expression constructs are delivered into cells, thereby inducing the production of further replicating hepatitis virus pseudoparticles. In this case, the retroviral genome is modified to express hepatitis virus glycoproteins E1 and E2 in place of the retroviral Env gene (encoding the retroviral glycoprotein). However, the gene encoding the core protein of the retrovirus remains unchanged. Further, for example, additional genes encoding marker genes or immunomodulators may also be expressed from the genome.

In one embodiment, the cell is an in vitro cell. In a preferred embodiment, the cells comprise mammalian cells. In a more preferred embodiment, the cells comprise human embryonic kidney cells (human embryonic KIDNEY CELLS).

In the present invention, the delivery of the expression construct may be carried out by any standard method well known to those skilled in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE glycosaminoglycan, calcium phosphate precipitation or the use of a gene gun.

In one embodiment, the expression construct further comprises a sequence for encoding a marker gene. In a preferred embodiment, the marker gene is a Green Fluorescent Protein (GFP) gene. The marker gene is intended for use in screening successfully transfected cells.

As used herein, the term "hepatitis C virus" (also known as hepatitis C virus, HEPATITIS C viruses, HCV) refers to any of a number of different genotypes and isolates of hepatitis C virus. Thus, "HCV" encompasses any of a variety of genotypes, subtypes or quasispecies of HCV, including, for example, genotypes 1, 2, 3, 4,6, 7, etc., and subtypes (e.g., 1a, 1b, 2a, 2b, 3a, 4c, etc.) and quasispecies. Representative HCV genotypes and isolates include: "Chiron" isolates HCV-1, H77, J6, con1, isolate 1, BK, EC1, EC10, HC-J2, HC-J5; HC-J6, HC-J7, HC-J8, HC-JT, HCT18, HCT27, HCV-476, HCV-KF, "Hunan", "Japanese", "Taiwan", TH, type 1a, type H77 type 1b, type 1c, type 1d, type 1e, type 1f, type 10, type 2a, type 2b, type 2c, type 2d, type 2f, type 3a, type 3b, type 3g, type 4a, type 4c, type 4d, type 4f, type 4H, type 4k, type 5a, type 6 and type 6 a.

The HCV genome encodes two membrane-associated envelope glycoproteins (E1 and E2), and they interact to form a non-covalent heterodimeric complex. HCV glycoproteins E1 and E2 are severely modified by N-linked glycosylation. The E1 protein is composed of 192 amino acids and contains 5 to 6N-glycosylation sites, depending on the HCV genotype. The E2 protein consists of 363 to 370 amino acids and contains 9-11N-glycosylation sites, depending on the HCV genotype.

In one embodiment, the viral antigen of HCV comprises glycoproteins E1 and E2 of HCV. The glycoproteins E1 and E2 of HCV are expressed on the surface of the HCV pseudoparticle. In a preferred embodiment, the HCV pseudoparticle has glycoproteins E1 and E2 of HCV.

As used herein, α2-3,6,8,9 neuraminidase a is a widely specific sialidase that cleaves linear and branched non-reducing terminal sialic acid residues from glycoproteins, glycopeptides and oligosaccharides. Thus, the present invention induces immunogenicity of HCV pseudoparticles by cleaving sialic acid on glycoproteins E1 and E2 of HCV pseudoparticles by α 2-3,6,8,9 neuraminidase a.

In another embodiment, in step (b), said HCV pseudoparticle is treated with said α2-3,6,8,9 neuraminidase a for 0.5 to 4 hours. In a preferred embodiment, in step (b), the HCV pseudoparticle is treated with α2-3,6,8,9 neuraminidase a for 0.5 to 3 hours. In a more preferred embodiment, in step (b), said HCV pseudoparticle is treated with said alpha 2-3,6,8,9 neuraminidase a for 1 to 2 hours.

In one embodiment, the method further comprises step (d) comprising mixing the HCV pseudoparticles of step (c) with a pharmaceutically acceptable carrier to produce an immunogenic composition. Still further, the HCV pseudoparticles of step (c) can be combined with the pharmaceutically acceptable carrier to produce an HCV vaccine.

The present invention also provides an immunogenic composition for eliciting an immune response to Hepatitis C Virus (HCV), wherein the immunogenic composition comprises an HCV pseudoparticle treated with alpha 2-3,6,8,9 neuraminidase a.

Furthermore, the immunogenic composition may optionally be combined with a suitable pharmaceutically acceptable carrier to enhance the protective effect of the HCV vaccine. In one embodiment, the HCV vaccine comprises the immunogenic composition and a pharmaceutically acceptable carrier.

The present invention further provides a method of eliciting an immune response to Hepatitis C Virus (HCV) in a subject comprising administering to the subject an effective dose of an HCV vaccine, wherein the HCV vaccine comprises an immunogenic composition, and the immunogenic composition comprises a 2-,6,8,9 neuraminidase a-treated HCV pseudoparticles.

In one embodiment, the subject is an animal, preferably a mammal, more preferably a human.

The present invention also provides a use of an immunogenic composition comprising HCV pseudoparticles treated with alpha 2-3, 6, 8, 9 neuraminidase a in the preparation of an HCV vaccine.

The vaccine of the present invention may be packaged into pharmaceutical or nutritional compositions or formulations along with additional active agents, carriers, vehicles, adjuvants, excipients or auxiliaries that will be appreciated by those skilled in the art after reading the present invention.

Adjuvants suitable for inclusion in the compositions of the present disclosure include those well known in the art, such as Freund's complete adjuvant (CFA), freund's incomplete adjuvant (IFA), squalene (squalene), squalane (squalane), alum and various oils, all of which are well known in the art and available from a variety of suppliers, such as North China (e.g., MF59 adjuvant).

In another embodiment, the vaccine comprises a pharmaceutically acceptable carrier. Thus, the immunogenic composition or vaccine of the invention further comprises a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds may also be added to the vaccine. The vaccine is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diamine tetraacetic acid; buffers, such as acetates, citrates or phosphates, and agents for adjusting tonicity, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, for example hydrochloric acid or sodium hydroxide. Parenteral formulations may be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

For parenteral administration, the immunogenic composition of the invention or vaccine thereof may be administered by intravenous, subcutaneous, intramuscular, intraperitoneal or intradermal injection, alone or in a composition further comprising a pharmaceutically acceptable carrier. For administration by injection, the immunogenic composition is preferably used in a solution of sterile aqueous vehicle, which may also contain other solutes, such as buffers or preservatives and sufficient pharmaceutically acceptable salts or glucose to render the solution isotonic. The immunogenic compositions of the invention may be obtained in the form of therapeutically acceptable salts well known in the art.

The vaccine may also be administered orally. Oral compositions typically include an inert diluent or an edible carrier. For the purposes of oral therapeutic administration, the active compounds may be combined with excipients and used in the form of tablets, dragees or capsules, such as gelatine capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients or compounds of similar nature: binders, such as microcrystalline cellulose, gum tragacanth or gelatin; excipients, for example starch or lactose; disintegrants, for example alginic acid, primogel or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

Systemic administration may also be transmucosal or transdermal. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels or creams as known in the art. The compounds may also be formulated in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect and associated with the desired pharmaceutical carrier.

It will be further appreciated that the amount of the immunogenic composition or vaccine of the invention that can be used to treat or prevent HCV will vary depending on the route of administration, the nature of the disorder being treated, and the age and condition of the subject, and will ultimately be at the discretion of the attendant physician.

The concentration of immunogen contained in the vaccine is that amount which will elicit an immune response without significant adverse side effects. The amount will vary depending on the immunogen used and the type and amount of adjuvant contained in the vaccine. Typically, the vaccine will comprise the immunogen in an amount of from about 0.1 to about 1000 μg/ml, preferably from about 0.2 to about 100 μg/ml, more preferably from about 0.5 μg to about 10 μg/ml. Following initial vaccination, the vaccinated subject may receive one or several booster immunizations at appropriate intervals thereafter.

Those of skill in the art will appreciate that the present disclosure extends from treatment to prevent established infections or symptoms. The immunogenic compositions and vaccines of the present invention may be administered as therapeutic or prophylactic. Treatment is preferably initiated prior to or at the time of infection or at the time of exposure of the mammal to HCV infection and continued until the virus is no longer present. However, treatment may also be initiated after infection, after exposure of the mammal to HCV infection, or after symptoms of the established infection.

Thus, when the immunogenic compositions of the invention are administered to a subject in need thereof, an immune response (e.g., a cellular immune response) is elicited in the subject against one or more HCV genotypes.

Drawings

FIG. 1 shows a transfer vector construct for producing HCV pseudo particles.

FIG. 2 shows the electrophoretic analysis of purified plasmids in 0.7% agarose gel. The left-hand numerical value of the figure is the Molecular Weight (MW) label (in kb). M: marking （marker）.A：phCMV-cE1E2(1a),MW = 7,754 bp.B：CMV-Gag-Pol MLV,MW = 11,984 bp.C：CMV-GFP MLV,MW = 6,309 bp.

FIG. 3 shows HEK293T post-transfection with intense green fluorescence.

FIG. 4 shows the neutralizing capacity of serum from HCV _Neura-A immunized mice. Serum from HCV _Neura-A immunized mice showed potent neutralizing activity (suggesting infection inhibition performance) compared to mice of the control group (saline injected). However, no significant inhibition was observed with serum from HCV _pp immunized mice. Female Balb/c mice (control/experimental group number of groups of 10) 6-8 weeks old were immunized by intraperitoneal injection. Each mouse was inoculated with 5. Mu.L of physiological saline (for control group), 5. Mu.g of HCV _pp or HCV _Neura-A with Freund's adjuvant. Priming (priming) on day 1, boosting (boosting) on days 14 and 28; blood was collected on days 1, 14, 28 and 42. Data are expressed as mean ± standard error of mean, number 10.* Representing p < <0.05, ns represents no significant statistical difference from the control group.

Detailed Description

This invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. The described embodiments should not be used to limit the scope of the invention as described in the claims.

Materials and methods

I. Reagent(s)

Alpha 2-3,6,8,9 neuraminidase A (Neura-A) was purchased from NEW ENGLAND Biolabs (Ipswich, mass., USA). DMEM, fetal bovine serum, streptomycin and penicillin were purchased from GIBCO BRL (Gaithersburg, md, USA). Freund's complete/incomplete adjuvant was purchased from SIGMA ALDRICH, inc. (St. Louis, mo, USA). Mag4C LV kit was purchased from OZ Biosciences (Av. De Luminy, marseille, france). Macrosep advanced centrifuge apparatus, MWCO 100K, available from Pall Corporation (Port Washington, new York, USA). TransIT-LT1 transfection reagent was purchased from Mirus, inc. (Madison, wis., USA).

II cell culture

HEK293T and huh7.5 cells were cultured in DMEM medium (Gibco) supplemented with 10% (v/v) fetal bovine serum, 10U/mL penicillin, 10 μg/mL streptomycin and 0.25 μg/mL amphotericin (amphotericin) B and at 37 ℃ and 5% CO ₂.

III production of HCV pseudo particle (HCV _pp)

The E1E2 and retroviral expression constructs (fig. 1) used to produce HCV pseudoparticles (HCV _pp) were provided by b. Bartosh doctor and f.l. Cosset doctor (INSERM U758, lyon, france) friends. HCV _pp is prepared as follows (including HCV E1E2 expression, packaging and transfer vectors).

III.1 packaging of transfer vector constructs

FIG. 1 shows CMV-Gag-Pol murine leukemia virus (murine leukemia virus, MLV) packaging constructs for encoding the MLV Gag and Pol genes, as well as MLV-GFP plasmids for encoding MLV-based transfer vectors containing CMV-GFP internal transcription units. The system exploits two well-documented properties of retroviruses, namely the ability to incorporate exogenous glycoproteins and the ability to integrate and express marker genes from non-replicating viral particles, resulting in a specific, rapid and reliable in vitro infection assay based on unmodified E1 and E2 HCV glycoproteins exhibited by pseudoparticles. HCV pseudoparticles (HCV _pp) are generated by assembling these full-length, unmodified E1 and E2 glycoproteins onto an MLV-derived retroviral core protein. Retroviruses were chosen as the platform for assembly of HCV _pp because their core can contain a variety of different cellular and viral glycoproteins, and because they can readily package and integrate gene markers into the DNA of infected cells. Immunoblot analysis of transfected cells showed that the structural components of the pseudo-particles were easily detected at the expected molecular weight; namely, E1 is about 30 kD, E2 is about 60 kD, and VSV-G is about 60 kD. Thus, this system describes for the first time the formation of highly infectious HCV pseudoparticles, and these pseudoparticles may share early cell entry properties with the parent HCV.

III.2 preparation and purification of plasmids

Plasmids phCMV-cE1E2 (1 a), CMV-Gag-Pol MLV and CMV-GFP MLV were propagated in E.coli, isolated and purified by equilibrium centrifugation in cesium chloride (CsCl) -ethidium bromide gradients (for production of HCV _pp by transfection into HEK293T cells).

III.3 production of naturally complete glycosylated pseudoparticles (HCV _pp)

To produce HCV _pp with native fully glycosylated E1E2, human embryonic kidney cells (HEK 293T), i.e., E1E2 glycoprotein (phCMV-cE 1E2 (1 a)), retroviral core protein (CMV-Gag-Pol MLV) and a packagable GFP-containing retroviral transfer vector (CMV-GFP MLV) were transfected by an expression vector for encoding the viral component. The sequences published by researchers on the gene bank (Genbank) (Genbank accession number AY 734972.1) for encoding E1 and E2 glycoproteins from HCV type 1a are SEQ ID NO:1. thus, the invention will have the sequence of SEQ ID NO:1 into an expression vector for expression of glycoproteins E1 and E2.

Briefly, DNA of Gag-Pol packaging construct (12 μg), transfer vector construct (12 μg) and glycoprotein expression construct (4 μg) was transfected into 2.5×10 ⁶ HEK293T cells seeded on a 10 cm plate the previous day, using TransIT-LT1 transfection reagent from Mirus, inc. (Madison, wis., USA) according to the manufacturer's guidelines. 16 hours after transfection, the medium was changed (8 mL/plate). 48 hours after transfection, the supernatant containing the pseudoparticles was collected, filtered through a 0.45 μm pore size membrane (for infection of huh7.5 cells) and concentrated 20-fold using a Macrosep Advance centrifugation device, MWCO 100K.

Test vaccine production (HCV _pp and HCV _Neura-A)

For the test vaccine HCV _Neura-A, alpha 2-3,6,8,9 neuraminidase a was used to cleave off native fully glycosylated HCV _pp, which was performed as follows: (1) 10 μg HCV _pp and water were mixed in a total reaction volume of 90 μl; (2) 10 mu L GlycoBuffer (10X) was added to give a total reaction volume of 100. Mu.L; (3) adding 10. Mu.L of alpha.2-3,6,8,9 neuraminidase A; and (4) incubation at 37℃for 1 hour.

For control experiments using native fully glycosylated HCV _pp as vaccine, HCV _pp was treated in the same way as described above, but without the addition of α2-3, 6, 8, 9 neuraminidase a.

Finally, the product was purified by a Mag4C LV magnetic nanoparticle kit from OZ Biosciences according to the manufacturer's guidelines procedure.

V. testing vaccine (HCV _pp and HCV _Neura-A) for immunogenicity

Glycosylation is known to affect protein folding and protein function. Glycans associated with viral membranes also play an important role in masking neutralizing epitopes and regulating the overall immunogenicity of viral particles. Such high degree of glycosylation on E1E2 suggests that these glycans can limit the immunogenicity of HCV envelope proteins and limit the binding of certain antibodies to epitopes on their virion surfaces, as observed for Human Immunodeficiency Virus (HIV) gp 120.

Furthermore, previous studies on influenza demonstrated that the mono-glycosylated hemagglutinin showed a similar secondary structure and better binding affinity to the host receptor compared to the fully glycosylated counterpart. Furthermore, asn of a single GlcNAc residue is the smallest component of N-glycans required for glycoprotein folding and stabilization.

To investigate the effect of deglycosylation with alpha 2-3,6,8,9 neuraminidase a on HCV _pp, BALB/c mice were immunized with saline (as control), HCV _pp or HCV _Neura-A.

V.1 in vivo animal studies

Female BALB/c,6-8 week old, purchased from BioLASCO (BioLASCO, taibei, taiwan, china) and maintained under conditions consistent with relevant guidelines and regulations for the care and use of laboratory animals at the university of Chinese medicine in Taiwan, china.

30 Mice were randomly and equally divided into 3 groups for evaluation of immunogenicity and toxicity of the test vaccines HCV _pp and HCV _Neura-A.

On days 1, 14 and 28, mice were given antisera by intraperitoneal injection of 5 μg HCV _pp、HCV_Neura-A or saline (for control) and an equal volume of freund's adjuvant. Antisera were taken on days 1, 14, 28 and 42. All serum samples were stored at-80 ℃ after collection.

Antisera from each group were compared and their ability to bind to native fully glycosylated HCV _pp (shown by infection inhibitory activity) was analyzed using a neutralization assay.

V.2. neutralization assay

To evaluate the neutralizing activity of serum from immunized mice, serum from control group, HCV _pp immunized mice and HCV _Neura-A immunized mice will be used for the study.

Huh7.5 cells were pre-seeded into 12 well cell culture plates at a density of 1 x 10 ⁵ per well. The next day, HCV _pp supernatants (225 μl/well) were combined with the samples from the control group, HCV _pp immunized mice and HCV _Neura-A at 1: an equal volume of antisera after 2 dilutions was incubated for 1 hour at 37 ℃. The mixture was then added to each well. After incubation at 37 ℃ for 3 hours, the supernatant was replaced with fresh medium and incubated at 37 ℃ for 72 hours. The entry of HCV was confirmed by the percentage of GFP positive cells measured by a Countess II FL automated cell counter (ThermoFisher Scientific, USA).

VI statistical analysis

Data are expressed as mean ± standard error. Evaluation of statistical significance was confirmed by single factor variance analysis (ANOVA) and p-values were calculated using SPSS 16.0 software. p values less than 0.05 are considered statistically significant.

Results

Preparation and purification of plasmids

Plasmids phCMV-cE1E2 (1 a), CMV-Gag-Pol MLV and CMV-GFP MLV were propagated in E.coli, isolated and purified by equilibrium centrifugation in a CsCl-ethidium bromide gradient. Purity was checked by 0.7% agarose gel electrophoresis as shown in fig. 2.

Production of naturally complete glycosylated pseudoparticles (HCV _pp)

To produce HCV _pp with native fully glycosylated E1E2, the expression vector encoding the viral component was transfected into human embryonic kidney cells (HEK 293T), i.e., the E1E2 glycoprotein, the retroviral core protein, and a packagable GFP-containing retroviral transfer vector. Finally, successfully transfected cells with intense green fluorescence were generated (fig. 3).

Immunogenicity in Balb/c mice: protective ability of serum of HCV _Neura-A immunized mice

To test the function of the test vaccines HCV _pp and HCV _Neura-A, neutralization assays were used to test all samples from the control group, HCV _pp immunized mice and HCV _Neura-A immunized mice for their ability to inhibit HCV _pp infection with huh7.5 cells.

The main correlation of the efficacy of the test vaccine is the extent of neutralizing antibodies (NtAb), which are shown as infection-inhibiting activity in the neutralization assay using huh7.5 cells. The neutralizing activity of the serum of saline-immunized mice and the serum of HCV _pp -immunized mice or HCV _Neura-A -immunized mice was compared with each other. Vaccine toxicity was also assessed together.

The present inventors found that all 10 serum samples from HCV _Neura-A immunized mice significantly inhibited the infectious activity of HCV _pp, suggesting an effective neutralizing capacity as well as protective capacity, compared to the control group.

For HCV _Neura-A immunized mice, the extent of infection inhibition correlates with priming and boosting time. As shown in fig. 4, a significant degree of inhibition of infection was detected 14 days after priming (74.69% ± 12.53) compared to the mice of the control group. After priming on day 1 and boosting on day 14, the neutralization activity reached a high level (53.87% ± 7.96) on day 28 and remained high (49.09±8.17) to day 42 (second boosting on day 28), suggesting that the neutralization activity (and specific protection against HCV _pp infection NtAb) increased in the same manner.

However, no significant inhibition of infection was observed for serum from naturally fully glycosylated HCV _pp immunized mice. As shown in fig. 4, the infection inhibition rate detected 14 days after priming was lower (96.47% ± 15.26) than that of the mice in the control group. The neutralization activity was boosted on days 1 and 14 after priming, kept low on day 28 (95.57% ± 14.17), and remained low (96.43% ± 15.69) to day 42 (second boosting on day 28).

Toxicity of

The test vaccine was subjected to a 6 week toxicology kinetics study. As described above, a total of 30 mice were randomly allocated to 3 groups (10 animals/group), including saline (control group), HCV _pp groups, and HCV _Neura-A groups, and were intraperitoneally injected on days 1, 14, and 28.

Under the conditions of the present invention, no morbidity or mortality was found; no adverse reactions, weight loss or other significant systemic toxicity were found in clinical observations during dosing and at the end of the 2-week recovery period for all animals.

Those skilled in the art will understand the concepts described above as descriptions of methods for delivering deposited application information. Those skilled in the art will recognize that these are merely illustrative and that many equivalents are possible.

Claims (7)

1. A method of preparing an immunogenic composition comprising a viral antigen of Hepatitis C Virus (HCV), comprising: (a) providing HCV pseudoparticles; (b) Treating the HCV pseudoparticle with α2-3,6,8,9 neuraminidase a to remove sialic acid on the surface of the HCV pseudoparticle; and (c) isolating the HCV pseudoparticles produced in step (b) to produce the immunogenic composition.

2. The method of claim 1, wherein the HCV pseudoparticle has glycoproteins E1 and E2 of HCV.

3. The method of claim 1, wherein in step (b), the HCV pseudoparticle is treated with the alpha 2-3,6,8,9 neuraminidase a for 0.5 to 4 hours.

4. The method of claim 1, further comprising step (d) comprising mixing the HCV pseudoparticles of step (c) with a pharmaceutically acceptable carrier to produce the immunogenic composition.

5. An immunogenic composition for eliciting an immune response to hepatitis C virus, wherein the immunogenic composition is prepared by the method of claim 1.

6. Use of an immunogenic composition for the preparation of a hepatitis C virus vaccine, wherein the immunogenic composition is prepared by the method of claim 1.

7. The use according to claim 6, wherein the hepatitis C virus vaccine is in a dosage range of 0.1-1000 μg/ml.