TWI844199B - Immunogenic compositions of hepatitis c virus and uses thereof - Google Patents

Abstract

The present invention provides a method for preparing an immunogenic composition, which comprises treating HCV pseudoparticles with α2-3,6,8,9 neuraminidase A to generate the immunogenic composition. In addition, the immunogenic compositions are made in vaccines for eliciting an immune response to HCV in a subject.

Description

Immunogenic compositions of hepatitis C virus and their uses

The present invention provides an immunogenic composition comprising hepatitis C virus (HCV) pseudoparticles treated with α2-3,6,8,9-neuraminase A. In addition, the present invention further provides the use of an immunogenic composition for preparing an HCV vaccine, wherein the immunogenic composition comprises HCV pseudoparticles treated with α2-3,6,8,9-neuraminase A.

Hepatitis C virus (HCV) is the leading etiological agent of chronic liver disease, affecting approximately 170 million people worldwide. The majority (approximately 85%) of individuals infected with HCV become chronically infected and are at risk for developing cirrhosis and hepatocellular carcinoma. HCV is a member of the Flaviviridae family and is an enveloped virus containing a positive-stranded genomic RNA. HCV can be divided into six major genotypes and numerous subtypes based on genomic sequence similarity. The HCV RNA genome contains 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 approximately 3010 amino acids, which is co-translated and transcribed by host and viral proteases into structural proteins (C, E1, E2 and p7) and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B).

Viral attachment and entry, which represent the first interaction of the virus with host cells, are the primary targets of the adaptive humoral response. The viral proteins responsible for cell attachment and HCV entry are glycoproteins E1 and E2. The viral proteins are recognized as foreign by the host immune system and induce the production of antibodies. A small fraction of these antibodies exhibit antiviral activity in vitro and are defined as virus neutralizing antibodies. Neutralizing antibody responses often provide the first line of adaptive defense against infection by limiting viral spread. Therefore, the expression of these glycoproteins has important applications in vaccine discovery and drug targets. Therefore, understanding the genotypic and quasispecies variation in viral glycoprotein properties is important for understanding the structure-function relationship of the proteins.

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

Glycans associated with the viral envelope have a major role in masking neutralizing epitopes and modulating the overall immunogenicity of the viral particle. Viral proteins are typically glycosylated by one of three different mechanisms. These processes differ not only in the cellular enzymes involved, but also in the different types of glycan structures produced. The glycans produced are referred to as N-linked, O-linked, or glycosylphosphatidyl inositol (GPI) anchored. In the N-linked glycosylation process, glycan chains are added to the viral protein via an asparagine residue. N-linked glycosylation occurs at sites in the protein where the consensus amino acid sequence Asn-X-Ser/Thr is present. This is by far the most common form of glycosylation of viral proteins, and they undergo N-linked glycosylation in a process similar to that which occurs on cellular glycoproteins.

Most glycosylation sites on the HCV envelope glycoproteins are conserved, and certain glycans associated with these proteins have been shown to play important roles in protein folding and HCV entry. Such highly expressed glycosylation suggests that these glycans may limit the immunogenicity of the HCV envelope protein and restrict the binding of certain antibodies to their epitopes on the virion surface, as has been observed for human immunodeficiency virus (HIV) gp120. At least three glycans on E2 (E2N1, E2N6, and E2N11) have been reported to reduce the sensitivity of the HCV _pp to antibody neutralization, and these glycans also reduce access of CD81 to its E2 binding site. In contrast, there is no evidence that the N-linked glycans of E1 contribute to masking of neutralizing epitopes. These data suggest that glycans E2N1, E2N6, and E2N11 are accessible to the binding site of CD81 and modulate the binding of CD81 and neutralizing antibodies to E2. In short, this work indicates that this region is a major target for neutralizing antibodies and that HCV glycans help HCV evade humoral immune responses.

The discovery of anti-HCV compounds and the development of HCV vaccines have been severely hampered by the lack of cell culture replication systems. Since the late 1990s, the availability of subgenomic replicons that mimic the intracellular reactions leading to HCV genome replication has enabled the discovery of HCV protease and polymerase inhibitors, but not the study of HCV entry or entry inhibitors. More recently, the function of HCV E1E2 in cell attachment and entry, which recapitulates the entire HCV replication cycle, has been investigated using a retrovirus-based pseudoparticle (pp) assay, in which the infectivity of the retroviral particles is conferred by the HCV E1E2 envelope protein. These new experimental systems have enabled rapid advances in our understanding of how HCV glycoproteins E1 and E2 mediate receptor binding and viral entry. These systems have facilitated the discovery of a range of viral receptors. This HCV _pp assay has been particularly useful for functional analysis of HCV E1E2 derived from patients infected with different genotypes and viral subtypes, dissecting the role of key E2 receptors CD81 and SR-B1 in viral entry, and measuring the ability of antibodies and patient sera to neutralize infection of HCV _pp -targeted cells.

Recently, infectious pseudoparticles assembled by expressing unmodified HCV envelope glycoprotein on retroviral core particles have been successfully produced. HCV pseudoparticles (HCVpp) were produced by transfecting 293T cells with three expression vectors encoding the E1E2 polyprotein, the retroviral core protein, and a packaging retroviral-derived genome with a marker gene. Surprisingly, infectious pseudoparticles were produced in the absence of any modification of the HCV envelope glycoprotein. The data accumulated on these pseudotyped particles strongly suggest that they can mimic the early steps of HCV infection. Indeed, they show a preferential tropism for hepatocytes and they can be specifically neutralized by anti-E2 monoclonal antibodies as well as by sera from HCV infected patients. Therefore, HCV _pp represents the best tool currently available to study the functional HCV envelope glycoprotein and provides a model system to study HCV cellular entry. The development of HCVpp offers 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 descriptions have shown that the gradual appearance of relatively strong neutralization reactions is associated with a decrease in viremia.

The present invention provides a method for preparing an immunogenic composition, which comprises HCV pseudoparticles treated with α2-3,6,8,9-neuraminase A to produce the immunogenic composition. The present invention also provides an immunogenic composition comprising HCV pseudoparticles treated with α2-3,6,8,9-neuraminase A. In addition, the present invention further provides the use of an immunogenic composition for preparing an HCV vaccine, wherein the immunogenic composition comprises HCV pseudoparticles treated with α2-3,6,8,9-neuraminase A.

Virus attachment and entry, representing the first interaction between the virus and the host cell, are the main targets of the adaptive humoral response. The viral proteins responsible for cell attachment and entry of HCV are glycoproteins E1 and E2. The present invention proposes deglycosylation of E1 and E2 by glycosidase, such as α2-3,6,8,9 neuraminase A (a broadly specific glycosidase that can cleave sialic acid residues at the linear and branched non-reducing ends from glycoproteins, glycopeptides and oligosaccharides), which can not only improve the sensitivity of detecting neutralizing antibody activity, but also induce an immune response that recognizes higher titers of E1 and E2 variants. In order to study the immunogenicity and protective ability of HCV pseudoparticles treated with α2-3,6,8,9-neuraminase A, a mouse model was used. In the present invention, the ability of antiserum of mice immunized with HCV _Neura-A to inhibit the entry of HCV _pp into Huh7.5 cells (represented by the inhibition of the infection rate of HCV _pp to Huh7.5 cells) was tested. The present invention shows that compared with the control group, immunization with HCV _Neura-A can induce an effective systemic humoral response with neutralizing activity, thereby effectively inhibiting the infection rate of HCV _pp to Huh7.5 cells. The present invention indicates that antiserum of mice immunized with HCV _Neura-A can effectively neutralize HCV _pp , thereby inhibiting HCV _pp (carrying intact and unmodified envelope glycoproteins of the parent origin) from entering cells, thereby inhibiting the infection of Huh7.5 cells. Therefore, based on the results obtained from the above neutralization studies, deglycosylation of HCV _pp can be used to design effective vaccines against HCV infection.

The strategy of the present invention (hydrolyzing sialic acid residues on HCV _pp glycoproteins via α2-3,6,8,9 neuraminase A) opens up a new direction for vaccine design and can be used together with other different vaccine strategies to promote the development of anti-HCV vaccines.

The term "a" or "an" herein is used to describe the elements and components of the present invention. The term is used only for convenience of description and to give the basic idea of the present invention. The description should be understood to include one or at least one, and unless clearly stated otherwise, the singular also includes the plural. When used in conjunction with the word "comprising" in the scope of the patent application, the term "a" can mean one or more.

As used herein in the claims, the term "or" means "and/or" unless expressly indicated to mean only other alternatives or unless the other alternatives are mutually exclusive.

The present invention provides a method for preparing an immunogenic composition comprising viral antigens of hepatitis C virus (HCV), comprising: (a) providing HCV pseudoparticles (HCV _pp ); (b) treating the HCV pseudoparticles with α2-3,6,8,9-neuraminase A to remove sialic acid on the surface of the HCV pseudoparticles; and (c) separating the HCV pseudoparticles produced in step (b) to produce the immunogenic composition.

In the present invention, the method for preparing the HCV pseudoparticles comprises: (1) delivering an expression construct into a cell, wherein the expression construct comprises a sequence for encoding HCV glycoproteins E1 and E2, retrovirus core protein and retrovirus-derived genome; and (2) isolating the HCV pseudoparticles produced by the cell.

In one embodiment, the sequence encoding HCV glycoproteins E1 and E2 comprises a sequence having SEQ ID NO: 1.

In the method for preparing the HCV pseudoparticles, three independent expression constructs can be designed, which contain three nucleic acid sequences for encoding the retroviral core, the genome and the hepatitis virus glycoprotein; or, a construct containing different nucleic acid sequences can be designed. Such expression constructs are delivered to cells, thereby inducing the production of further replicated hepatitis virus pseudoparticles. In this case, the retroviral genome is modified to express the hepatitis virus glycoproteins E1 and E2 to replace the retroviral Env gene (encoding the retroviral glycoprotein). However, the gene encoding the retroviral core protein remains unchanged. Furthermore, for example, additional genes encoding marker genes or immunomodulators can also be expressed from the genome.

In one embodiment, the cell is an in vitro cell. In a preferred embodiment, the cell comprises a mammalian cell. In a more preferred embodiment, the cell comprises a human embryonic kidney cell.

In the present invention, the delivery of the expression construct can be carried out by any standard method known to those skilled in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE glucan, 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 green fluorescent protein (GFP). The purpose of the marker gene is to screen successfully transfected cells.

As used herein, the term "hepatitis C virus" ("HCV") refers to any of a variety 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, 4a, 4c, etc.) and quasispecies. Representative HCV genotypes and isolates include: "Chiron" isolate 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", "Japan", "Taiwan", TH, 1, 1a, H77 1b, 1c, 1d, 1e, 1f, 10, 2, 2a, 2b, 2c, 2d, 2f, 3, 3a, 3b, 3g, 4, 4a, 4c, 4d, 4f, 4h, 4k, 5, 5a, 6, and 6a.

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

In one embodiment, the HCV viral antigens include HCV glycoproteins E1 and E2. The HCV glycoproteins E1 and E2 are expressed on the surface of the HCV pseudoparticles. In a preferred embodiment, the HCV pseudoparticles have HCV glycoproteins E1 and E2.

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

In another embodiment, in step (b), the HCV pseudoparticles are treated with the α2-3,6,8,9-neuraminase A for 0.5 to 4 hours. In a preferred embodiment, in step (b), the HCV pseudoparticles are treated with the α2-3,6,8,9-neuraminase A for 0.5 to 3 hours. In a more preferred embodiment, in step (b), the HCV pseudoparticles are treated with the α2-3,6,8,9-neuraminase A for 1 to 2 hours.

In one embodiment, the method further comprises a step (d), which comprises mixing the HCV pseudoparticles of step (c) with a pharmaceutically acceptable carrier to produce the immunogenic composition. Furthermore, 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 inducing an immune response to hepatitis C virus (HCV), wherein the immunogenic composition comprises HCV pseudoparticles treated with α2-3,6,8,9-neuraminase A.

In addition, the immunogenic composition can be optionally 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 for inducing 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 an HCV pseudoparticle treated with α2-,6,8,9-neuraminase A.

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

The present invention also provides an immunogenic composition for use in preparing an HCV vaccine, wherein the immunogenic composition comprises an HCV pseudoparticle treated with α2-3, 6, 8, 9 neuraminidase A.

The vaccine of the present invention can be incorporated into a pharmaceutical or nutritional composition or preparation together with additional active agents, carriers, vehicles, adjuvants, excipients or auxiliary agents that can be identified by a person 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 complete Freund's adjuvant (CFA) not for use in humans, incomplete Freund's adjuvant (IFA), squalene, squalane, alum, and various oils, all of which are well known in the art and available from a variety of suppliers, such as Novartis (e.g., MF59 adjuvant).

In another embodiment, the vaccine comprises a pharmaceutically acceptable carrier. Therefore, the immunogenic composition or vaccine of the present 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 agents and absorption delaying agents, etc. that are compatible with the administration of the drug. Supplementary active compounds can 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, such as 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; antibacterial agents, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and agents for adjusting tonicity, such as sodium chloride or glucose. The pH value can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

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

The vaccine can also be administered orally. Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be combined with a formulation and used in the form of tablets, tablets or capsules, such as gelatin capsules. Oral compositions can also be prepared using fluid carriers for use as mouthwashes. Pharmaceutically compatible binders or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, tablets, etc. may contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as 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 flavorings such as mint, 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 may 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 are 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 individual to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier.

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

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

Those skilled in the art will appreciate that references herein extend from treatment to prevention as well as treatment of established infection or symptoms. The immunogenic compositions and vaccines of the invention may be administered therapeutically or prophylactically. Treatment preferably begins prior to or at the time of infection or upon exposure of the mammal to HCV infection and continues until the virus is no longer present. However, treatment may also begin after infection, after exposure of the mammal to HCV infection, or after symptoms of an established infection develop.

Therefore, when the immunogenic composition of the present invention is administered to an individual in need thereof, an immune response (e.g., a cellular immune response) against one or more HCV genotypes is induced in the individual.

Figure 1 shows the transfer vector construct used to produce HCV pseudoparticles.

Figure 2 shows the electrophoresis analysis of purified plasmids in 0.7% agarose gel. The values on the left side of the figure are molecular weight (MW) markers (in kb). M: marker. A: phCMV-cE1E2(1a), MW=7,754bp. B: CMV-Gag-Pol MLV, MW=11,984bp. C: CMV-GFP MLV, MW=6,309bp.

Figure 3 shows transfected HEK293T with strong green fluorescence.

Figure 4 shows the neutralizing ability of sera from mice immunized with HCV _Neura-A . Sera from mice immunized with HCV _Neura-A showed effective neutralizing activity (suggesting infection inhibition) compared with mice in the control group (injected with saline). However, no significant inhibitory effect was observed with sera from mice immunized with HCV _pp . Female Balb/c mice aged 6-8 weeks (10 in each control/experimental group) were immunized by intraperitoneal injection. Each mouse was inoculated with 5 μL saline (for the control group), 5 μg HCV _pp or HCV _Neura-A with Freund's adjuvant. Priming was performed on day 1, boosting on days 14 and 28; blood was collected on days 1, 14, 28 and 42. Data are presented as mean ± standard error of the mean, with a value of 10. * indicates p < 0.05, and ns indicates no significant statistical difference compared with the control group.

The present invention may be embodied in many different forms and should not be construed as limited to the embodiments described herein. The described embodiments are not intended to limit the scope of the invention as described in the claims.

Materials and methods

I. Test reagent

α2-3,6,8,9-neuraminase A (Neura-A) was purchased from New England Biolabs (Ipswich, MA, 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 Advance centrifuge, MWCO 100K, was purchased from Pall Corporation (Port Washington, New York, USA). TransIT-LT1 transfection reagent was purchased from Mirus, Inc. (Madison, WI, USA).

II. Cell culture

HEK293T and Huh7.5 cells were cultured in DMEM medium (Gibco) supplemented with 10% (v/v) fetal bovine serum, 10 U/mL penicillin, 10 μg/mL streptomycin, and 0.25 μg/mL amphotericin B at 37°C and 5% CO ₂ .

III. Production of HCV Pseudoparticles (HCV _pp )

E1E2 and retroviral expression constructs for the production of HCV pseudoparticles (HCV _pp ) ( FIG1 ) were kindly provided by Dr. B. Bartosh and Dr. FL Cosset (INSERM U758, Lyon, France). HCV pp was prepared as described below (including HCV E1E2 expression, packaging, and transfer vectors).

III.1. Packaging of transfer vector constructs

Figure 1 shows the CMV-Gag-Pol murine leukemia virus (MLV) packaging construct encoding the MLV gag and pol genes and the MLV-GFP plasmid encoding the MLV-based transfer vector containing the CMV-GFP internal transcriptional unit. This system exploits two well-documented properties of retroviruses, the ability to incorporate foreign glycoproteins and the ability to integrate and express marker genes from replication-incompetent viral particles, resulting in a specific, rapid, and reliable in vitro infection assay based on pseudoparticle display of unmodified E1 and E2 HCV glycoproteins. HCV pseudoparticles (HCV _pp ) are generated by assembling these full-length, unmodified E1 and E2 glycoproteins onto MLV-derived retroviral core proteins. Retroviruses were chosen as platforms for the assembly of HCV _pp because their cores can contain a variety of different cellular and viral glycoproteins and because they can easily package and integrate genetic markers into the DNA of infected cells. Immunoblot analysis of transfected cells showed that the structural components of the pseudoparticles were readily detectable at the expected molecular weights; i.e., approximately 30 kD for E1, approximately 60 kD for E2, and approximately 60 kD for VSV-G. Thus, the formation of highly infectious HCV pseudoparticles has been described for the first time in this system, and these pseudoparticles may share early cell entry properties with the parental HCV.

III.2. Preparation and purification of plasmids

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

III.3. Production of native fully glycosylated pseudoparticles (HCV _pp )

To generate HCV _pp with native fully glycosylated E1E2, human embryonic kidney cells (HEK293T) were transfected with expression vectors encoding viral components, namely E1E2 glycoprotein (phCMV-cE1E2(1a)), retroviral core protein (CMV-Gag-Pol MLV) and a packageable retroviral transfer vector containing GFP (CMV-GFP MLV). The sequence encoding E1 and E2 glycoproteins from HCV type 1a published by researchers on Genbank (Genbank accession number AY734972.1) is SEQ ID NO: 1. Therefore, the present invention incorporates the sequence of SEQ ID No: 1 into the expression vector to express glycoproteins E1 and E2.

Briefly, DNA of Gag-Pol packaging construct (12 μg), transfer vector construct (12 μg), and glycoprotein expression construct (4 μg) were transfected into 2.5 x 10 ⁶ HEK293T cells seeded in 10 cm dishes the day before using TransIT-LT1 transfection reagent from Mirus, Inc. (Madison, WI, USA) according to the manufacturer's protocol. 16 hours after transfection, the medium was changed (8 mL/dish). 48 h after transfection, the supernatant containing 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 centrifuge, MWCO 100K.

IV. Testing vaccine production (HCV _pp and HCV _Neura-A )

For the test vaccine HCV _Neura-A , α2-3,6,8,9-neuraminase A was used to cleave native fully glycosylated HCV _pp , and the steps were as follows: (1) 10 μg of HCV _pp and water were mixed in a total reaction volume of 90 μL; (2) 10 μL of GlycoBuffer 1 (10X) was added to make the total reaction volume 100 μL; (3) 10 μL of α2-3,6,8,9-neuraminase A was added; and (4) incubated at 37°C for 1 hour.

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

Finally, the product was purified using the Mag4C LV Magnetic Nanoparticle Kit from OZ Biosciences according to the manufacturer’s guidelines.

V. Testing the immunogenicity of vaccines (HCV _pp and HCV _Neura-A )

Glycosylation is known to affect protein folding as well as protein function. Glycans associated with the viral envelope also play an important role in masking neutralizing epitopes and modulating the overall immunogenicity of the viral particles. Such a high degree of glycosylation on E1E2 suggests that these glycans can limit the immunogenicity of the HCV envelope protein and restrict the binding of certain antibodies to epitopes on the surface of its viral particles, as has been observed for human immunodeficiency virus (HIV) gp120.

Furthermore, previous studies on influenza have demonstrated that monoglycosylated hemagglutinins display similar secondary structures and better binding affinity to host receptors than fully glycosylated counterparts. Furthermore, Asn with a single GlcNAc residue is the minimal component of the N-glycan required for glycoprotein folding and stability.

To investigate the effect of deglycosylation of HCV _pp by α2-3,6,8,9-neuraminase A, BALB/c mice were immunized with saline (as a control), HCV _pp , or HCV _Neura-A .

V.1. In vivo animal studies

Female BALB/c, 6–8 weeks old, were purchased from BioLASCO (BioLASCO, Taipei, Taiwan) and maintained under conditions that complied with the relevant guidelines and regulations for the care and use of experimental animals of China Medical University.

Thirty mice were randomly and equally divided into three groups for evaluation of the immunogenicity and toxicity of the test vaccines HCV _pp and HCV _Neura-A .

Antisera were generated by intraperitoneal injection of 5 μg HCV _pp , HCV _Neura-A or saline (for control group) and an equal volume of Freund's adjuvant on days 1, 14 and 28. Antisera were obtained on days 1, 14, 28 and 42. All serum samples were stored at -80°C after collection.

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

V.2. Neutralization test

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

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

VI. Statistical analysis

Data are presented as mean ± standard error. One-way analysis of variance (ANOVA) was used to determine the statistical significance, and p-values were calculated using SPSS 16.0 software. A p-value less than 0.05 was considered statistically significant.

result

Preparation and purification of plasmids

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

Production of natural fully glycosylated pseudoparticles (HCV _pp )

To generate HCV _pp with native fully glycosylated E1E2, human embryonic kidney cells (HEK293T) were transfected with expression vectors encoding viral components, namely E1E2 glycoprotein, retroviral core protein, and a packageable retroviral transfer vector containing GFP. Finally, successfully transfected cells with strong green fluorescence were produced (Figure 3).

Immunogenicity in Balb/c mice: Protective capacity of sera from mice immunized with HCV _Neura-A

To test the functionality of the test vaccines HCV _pp and HCV _Neura-A , all samples from control, HCV _pp- immunized mice, and HCV _Neura-A- immunized mice were tested for their ability to inhibit HCV _pp infection of Huh7.5 cells using a neutralization assay.

The main correlate of vaccine efficacy is the degree of neutralizing antibodies (NtAb), expressed as infection inhibitory activity in a neutralization assay using Huh7.5 cells. The neutralizing activity of sera from saline-immunized mice and sera from HCV _pp- immunized mice or HCV _Neura-A- immunized mice was compared with each other. The toxicity of the vaccine was also evaluated.

The present invention found that all 10 serum samples from HCV _Neura-A immunized mice significantly inhibited the infection activity of HCV _pp compared with the control group, suggesting effective neutralization and protection ability.

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

However, no significant inhibition of infection was observed for sera from mice immunized with native fully glycosylated HCV _pp . As shown in Figure 4, the infection inhibition rate detected 14 days after priming was lower (96.47% ± 15.26) compared with the control group of mice. After boosting on days 1 and 14 after priming, the neutralizing activity remained low on day 28 (95.57% ± 14.17) and remained low (96.43% ± 15.69) until day 42 (second boost on day 28).

Toxicity

A 6-week toxicokinetics study of the test vaccine was conducted. A total of 30 mice were randomly assigned to 3 groups (10 animals/group), including saline (control), HCV _pp , and HCV _Neura-A , and injected intraperitoneally on days 1, 14, and 28, as described above.

Under the conditions of the present invention, no illness or death was found; all animals were observed to have no abnormal reactions, weight loss or other obvious systemic toxicity during the administration period and at the end of the 2-week recovery period.

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

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Claims (7)

A method for preparing an immunogenic composition comprising viral antigens of hepatitis C virus (HCV), comprising: (a) providing HCV pseudoparticles; (b) treating the HCV pseudoparticles with α2-3,6,8,9-neuraminase A to remove sialic acid on the surface of the HCV pseudoparticles; and (c) separating the HCV pseudoparticles produced in step (b) to produce the immunogenic composition. The method as described in claim 1, wherein the HCV pseudoparticles have HCV glycoproteins E1 and E2. The method as claimed in claim 1, wherein in step (b), the HCV pseudoparticles are treated with the α2-3,6,8,9-neuraminidase A for 0.5 to 4 hours. The method as described in claim 1 further comprises a step (d), which comprises mixing the HCV pseudoparticles of step (c) with a pharmaceutically acceptable carrier to produce the immunogenic composition. An immunogenic composition for inducing an immune response to hepatitis C virus, wherein the immunogenic composition is prepared by the method of claim 1. An immunogenic composition for use in preparing a hepatitis C virus vaccine, wherein the immunogenic composition is prepared by the method of claim 1. The use as described in claim 6, wherein the dosage range of the hepatitis C virus vaccine is 0.1-1000μg/ml.