Hepatitis C virus E1 and modified E2 delivered from mRNA vaccines induce protective immunity

Generation of mRNA-LNP vaccines encoding HCV envelope glycoproteins

Codon-optimized luciferase and HCV (genotype 1a/H77C) envelope glycoproteins sE1 (aa 193–351), sE2 (aa 386–660), and modified sE2_F442NYT A specific sequence was constructed. HCV sE2 and sE2_F442NYT Following PNGaseF treatment at the Washington University Proteomics Shared Resource in St. Louis (WU-PSR), protein samples were examined and compared for glycosylation sites inserted into F442.Glycosylation site at residue F442 of modified sE2_F442NYT Proteins were observed after constitutive deamidation of that residue and variable deamidation of other asparagine residues within its sequence. mRNA from HCV sE1, sE2, or sE2_F442NYT generated, purified, and encapsulated in LNPs for use as candidate vaccines in this study⁸.

codon-optimized luciferase HCV E1_193–351 and HCV E2_386–660 Specific sequences, with or without mutations at residues 442 and 444, were synthesized into mRNA, purified and encapsulated in LNPs for use in mouse immunization. An ethanol lipid mixture containing ionized cationic lipids, phosphatidylcholine, cholesterol, and polyethylene glycol lipids was rapidly mixed with an aqueous solution containing cellulose purified N1-mΨ with in vitro transcribed mRNA. LNPs loaded with mRNA were formulated using a total lipid concentration of 40 mM. RNA-loaded particles were characterized by size, surface charge, encapsulation efficiency, and endotoxin content and stored at −80 °C at an RNA concentration of 1 μg/μL (for loaded particles) and a total lipid concentration of 30 μg. /μL (both loaded and empty particles). The mRNA-LNPs had an average hydrodynamic diameter of ~80 nm, a polydispersity index of 0.02 to 0.06, and an encapsulation efficiency of ~95%. The LNP formulation used in this study is the property of Acuitas Therapeutics (US Patent 10,221,127). Single-use aliquots of vaccine preparations were used for immunizing mice.

Immunization of mice with mRNA-LNP vaccine and challenge infection with recombinant vaccinia virus

BALB/c mice (Jackson Lab) were divided into 5 groups (5 mice per group) and each group of mice was treated intramuscularly with 10 μg mRNA-LNP candidate vaccine as sE1/sE2, sE1, sE2, sE2. immunized to_F442NYT, sE1/sE2_F442NYT, or 2 vehicle controls at 2-week intervals. HCV vaccinia challenge as a surrogate model is useful for analyzing protective responses to challenge infection.^{8,20,twenty three,twenty four}Test bleeds from mice (3 days prior to immunization and 3 days prior to challenge infection) were analyzed. The use of a surrogate recombinant vaccinia virus (vv)/HCV challenge model in our study examined the biological relevance of T cell responses, rather than antibodies, as a function for protection from challenge infection.

Immunized mice were challenged intraperitoneally with live recombinant vaccinia virus expressing HCV E1-E2-NS2._134–966 or HCVE2-NS2-NS3_364–1619 (genotype1a/H77C), sacrificed 4 days after challenge infection to collect ovaries for further analysis. Ovaries were homogenized, freeze-thawed three times and centrifuged. Clarified supernatants were serially diluted for vaccinia virus titer determination by plaque formation assay on BSC-40 cell monolayers. After 3 days, plaques were stained with 1% crystal violet and counted.

ethics statement

All animal experiments were performed in accordance with relevant local, state, and federal regulations. The study was approved by the St. Louis University Institutional Animal Care and Use Committee (IACUC).

Cytokine quantification

Test bleeds from mice (3 days prior to immunization and 3 days prior to challenge infection) were analyzed. Serum cytokines IL-2 (Sigma, RAB0287), IFN-γ (R&D Systems, DY485), IL-4 (Biolegend, 431101), and IL-10 (Invitrogen, 88–7105–22) were measured from animals. Mice by ELISA using a commercial kit according to manufacturer’s instructions and dilution guidelines. Similarly, mouse IL-2 (Sigma, RAB0287), IFN-γ (R&D Systems, DY485), and granzyme B (R&D Systems, DY1865) were quantified from ovarian homogenates of mice after challenge infection with recombinant vaccinia virus. it was done.

HCV pseudoparticle neutralization assay

HCV pseudoparticles (HCVpp) were isolated from HEK293T cells using the HDM-Hgpm2-pRC-CMV-Rev1b-HDM-tat1b packaging vector, the Luciferase-IRES-ZsGreen plasmid (BEI Resources), and a plasmid expressing E1E2 from HCV. generated by co-transfection. Genotypes 1a (H77C), 1b (1b58), 3 (3.1.2), and 4(4.1.2) are available in our laboratory or Lipofectamine 3000 (Invitrogen L3000–008)^{twenty three}The supernatant containing HCVpp was harvested 72 hours after transfection and filtered through a nitrocellulose membrane with a pore size of 0.45 μm. For HCVpp neutralization test, 1.5 x 10^Four Huh7.5 cells were seeded per well in 24-well tissue culture plates and incubated overnight at 37 °C. The next day, HCVpp was mixed with or without different serial dilutions (1:100, 1:200, 1:400, 1:800, 1:1600, 1:2400, 1:3200) of immunized mouse sera and treated with Huh7. 5 1 h at 37 °C before adding to cells. After 72 h at 37 °C, cells were lysed with cell lysis buffer (EI53A, Promega) and 100 μl luciferase substrate (EI51A, Promega) was added to each well. Luciferase activity was measured in relative luminescence units (RLU) using a Glomax luminometer (Promega). The neutralization inhibitory concentration (IC50) for pseudotype infectivity was defined as a 50% or greater reduction in luciferase activity using the following formula:⁶Neutralization rate was calculated as:[1 − (RLU_mAb/RLU_untreated)]× 100, averaged from triplicate untreated control RLU values. Neutralizing activity was presented at dilutions of serum samples using lower and upper limits (0% and 100% inhibition).

Subclass- or isotype-specific immunoglobulin response

Nunc MaxiSorp ELISA plates were coated with 50 μl of 1 μg/ml purified HCV E1E2 protein (Chiron) in 0.1 M sodium bicarbonate buffer (pH 7.2) overnight at 4 °C. Wells were blocked with 2.5% BSA/PBS blocking buffer for 2 hours at 37 °C and washed 4 times with 0.01% Tween 20 in PBS. Mouse sera were serially diluted (1:50, 1:100, 1:200) in blocking buffer, added to the plate, incubated overnight at 4 °C, and washed. Different rabbit HRP-tagged anti-mouse immunoglobulins (subclasses: IgG, IgM, IgA, and isotypes: IgG1, IgG2a, IgG2b, IgG3) (Bio-Rad, Mouse Typer Isotyping Panel, 1722055) are added to appropriate wells and °C for 1 hour, followed by 4 washes. The HRP conjugate was added at 1:3000, incubated for 1 hour, and the wash cycle repeated. 100 μl of peroxidase substrate solution was then added and the reaction stopped with 2M sulfuric acid. Absorbance was measured at 450 nm using an ELISA plate reader (Tecan).

HCV envelope peptide-specific reactivity

Peptide-specific serum IgG binding reveals diverse genotype-specific conserved neutralizing epitopes in HCV H77 strains (NR4062 and NR4063, BEI Resources)^{9,Ten,12,13,14,15}A SARS-CoV-2 fusion domain peptide was used as an irrelevant control. Wells were coated with 500 ng of peptide and ELISA was performed using different dilutions of the above immune mouse sera (1:200, 1:400, 1:800).

statistical analysis

All data were analyzed using GraphPad Prism 7 software. Analysis of data for only two groups was performed utilizing the one-tailed Mann-Whitney U test or unpaired test. t-test. One-way ANOVA with Kruskal-Wallis test was used for multiple pairwise comparisons between groups. All data are expressed as mean ± SD (standard deviation of the mean). p< 0.05 was considered statistically significant.

Report overview

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