Broadly neutralizing immune responses against hepatitis C virus induced by vectored measles viruses and a recombinant envelope protein booster

Abstract

Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination.

Fig 1

Genome and growth characteristics of vectored MV expressing HCV proteins. (A) Diagram of the genomes of recombinant vectors. MV proteins are indicated by dark gray arrows, HCV proteins by inserts of light gray arrows. For details on construction, see Materials and Methods. The names of the recombinant MVs are indicated above the genome diagrams. (B) Time course of cell-associated (top) and cell-free virus production in Vero/hSLAM cells infected with MVvac2 (circles), MV-CE1E2 (squares), or MV-E1/Ft-E2/Ft (triangles). Viral titers are indicated on the vertical axes. These titers were measured at 12, 24, 36, 48, 72, and 96 h postinfection. For clarity, symbols were moved slightly sideways. Averages and standard deviations of three independent experiments are indicated.

Fig 2

Biochemical characterization of HCV proteins expressed by vectored MVs. (A) Expression of HCV core protein by vectored MV-CE1E2. Extracts from mock-, MVvac2-, MV-CE1E2-, or MV-E1/Ft-E2/Ft-infected cells were analyzed by immunoblotting. The reactivity of each antibody is indicated below each blot. (B) Immunoprecipitation of proteins produced by cells infected with MVvac2, MV-CE1E2, or MV-E1/Ft-E2/Ft at an MOI of 0.5. Proteins were labeled with [³⁵S]methionine 24 h postinfection and precipitated with the antibody indicated below the lanes. The positions of molecular mass standards are indicated on the left. (C) Immunoprecipitation of HCV envelope proteins using conformation-dependent monoclonal antibodies H53 and H60, as indicated below the lanes. (D) Glycosidase treatment. MV H and HCV E1 complexes immunoprecipitated from cells infected with MV-CE1E2 or MV-E1/Ft-E2/Ft were treated with endoglycosidase H (E) or PNGase-F (F) or left untreated (U). MV H (top panel) and HCV E1 glycoproteins (botttom panel) were detected by immunoblotting using specific antibodies. The asterisk beside E1 indicates an endoglycosidase H-resistant form. (E) Surface biotinylation analysis. Cells were infected with MVvac2, MV-CE1E2, or MV-E1/Ft-E2/Ft at an MOI of 0.1. Forty-eight hours later, cells were labeled with biotin and lysed. Biotin-labeled proteins were pulled down with streptavidin-agarose and analyzed by immunoblotting. Antibodies used for specific detection are indicated at the bottom.

Fig 3

Hybrid HCV glycoproteins are not incorporated into measles virus virions. MV particles or infected-cell lysates, as indicated below each blot, were analyzed using a polyclonal serum specific for the F cytoplasmic tail. F₀ and F₁, precursor and mature forms of the F protein, respectively.

Fig 4

Humoral immune response in genetically modified mice immunized with vectored MV expressing HCV structural proteins. Groups of 7 animals were immunized with two doses of MVvac2 (squares), CE1E2 (circles), or E1/Ft-E2/Ft (triangles). Each symbol represents one animal; a horizontal bar shows the average of the group. Sera were obtained either 4 weeks after the initial infection (4w) or 2 weeks after repeat infection (6w). All sera were assayed for MV neutralization (A; reciprocals of the titer), for reactivity against HCV E2 by ELISA (B; optical density at 450 nm), or for HCV neutralization using an H77-pseudotyped luciferase-expressing retrovirus (C; percent neutralization). In panel B, average readings for the background (−) and monoclonal antibody anti-E2 (+) are shown with gray and black bars, respectively. Even though most of the samples were anti-E2 seropositive as detected by ELISA after the second dose, one and three animals in the MV-CE1E2 and MV-E1/Ft-E2/Ft groups, respectively, had background level values. For panel C, two mice in each experimental group did not develop detectable levels of neutralizing antibodies.

Fig 5

Humoral immune responses in genetically modified mice immunized with vectored MVs and boosted with a single dose of E2 protein. Groups of 5 to 8 animals were immunized with two doses of MVvac2 (squares), CE1E2 (circles), or E1/Ft-E2/Ft (triangles) and boosted with 5 μg of E2 protein treated in the presence of Freund's complete adjuvant. Sera obtained 4 weeks after viral inoculation (4w, solid symbols) or 4 weeks after subsequent homologous protein boost (8w, empty symbols) were assayed for MV neutralization (A; reciprocals of the titer), reactivity against HCV E2 by ELISA (B; optical density at 450 nm), or HCV neutralization using an H77-pseudotyped luciferase-expressing retrovirus (C; percentage of neutralization normalized by MVvac2 readings). In panel B, average readings for the background (−) and a monoclonal antibody, anti-E2 (+), are shown with gray and black bars, respectively. Each symbol represents one animal; a horizontal bar indicates the group average.

Fig 6

Cross-neutralizing immune responses against cell culture-replicating HCV. Huh-7.5 cells were infected with an MOI of 0.1. The first and second sets of 3 columns show neutralization with control antibodies anti-CD81 and anti-E2. Levels of neutralization of chimeric HCVcc containing structural proteins are indicated as follows: H77 (genotype 1a) with black columns, Con1 (genotype 1b) with dark gray columns, and J6 (genotype 2a) with light gray columns. Other sets of columns show neutralization levels observed in sera of 5 mice primed with control MVvac2, 8 mice primed with MV-E1/Ft-E2/Ft, or 8 mice primed with MV-CE1E2. All mouse serum samples were diluted 1:200. At the bottom is shown the statistical significance of the differences observed between the groups, using the same color-coding of the chimeric HCVcc genotypes as for the neutralization assay.