Characterization of immune responses induced by intramuscular vaccination with DNA vaccines encoding measles virus hemagglutinin and/or fusion proteins

Abstract

Measles virus (MV) hemagglutinin (MV-H) and fusion (MV-F) proteins induce plaque reduction neutralizing (PRN) antibodies and cell-mediated immune responses that protect against clinical measles. DNA vaccines that encode MV-H and MV-F are being investigated as a new generation of measles vaccine to protect infants too young to receive currently licensed attenuated measles vaccines. However, it is unclear whether DNA vaccines encoding both MV-H and MV-F act synergistically to induce stronger immunity than immunization with plasmids encoding MV-H or MV-F alone. To address this question, we generated Sindbis virus-based pSINCP DNA vaccines that encode either MV-H or MV-F alone or bicistronic or fusion system vectors that encode both MV-H and MV-F (to mimic MV infection where both MV-H and MV-F proteins are expressed by the same mammalian cell). Mice immunized with DNA vaccine encoding MV-H alone developed significantly greater PRN titers than mice immunized with bicistronic constructs. Interestingly, the presence of MV-F in the bicistronic constructs stimulated serum MV-specific immunoglobulin G of reduced avidity. By contrast, mice immunized with bicistronic constructs induced equivalent or higher levels of MV-specific gamma interferon responses than mice immunized with DNA vaccine encoding MV-H alone. These data will help guide the design of DNA-based MV vaccines to be used early in life in a heterologous prime-boost strategy.

FIG. 1.

Schematic diagram of eukaryotic expression vectors. All constructs (not drawn to scale) were based on pSINCP. The pMSIN-H vector contains a full-length MV-H under the control of Sindbis virus subgenomic promoter (SubP). pMSIN-FL and pMSIN-FdU encode MV-F with or without its 5′ NTR, respectively. The bicistronic expression vectors, pMSINH-FdU and pMSINH-IRESFdU, encode MV-F under the control of SubP or the IRES element, respectively. The plasmid pMSINH-FdU(dC) contains MVF with mutations in its cleavage site (RRHKR to RRTSR at amino acids 108 to 112). The fusion construct, pMSINH-FdU(fu), encodes an in-frame fusion of MV-H, lacking its termination codon, with MV-F, lacking its signal sequence. Numbers in parentheses indicate the number of amino acids encoded by each DNA fragment.

FIG. 2.

Identification of the MV-H and MV-F proteins expressed in BHK-21 cells. Cell lysates were prepared from BHK-21 cells 48 h posttransfection with the different plasmid constructs. Cell lysates were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred, and the immunoblot was probed with a mouse monoclonal anti-MV-H antibody (A) or with anti-MV-F antibody (B). Sizes are indicated in kilodaltons.

FIG. 3.

Syncytium formation in Vero cells transiently expressing MV-H and/or MV-F protein. Vero cells were transfected with the various plasmid constructs as indicated. Cells were photographed 48 h after transfection by phase-contrast microscopy. Magnification, ×100.

FIG. 4.

Induction of MV-specific IgG antibody in mice immunized i.m. with DNA vaccines encoding MV-H and/or -F. Groups of BALB/c mice (n = 10) were immunized i.m. on days 0 and 28 with 100 μg of the various plasmid constructs as depicted on the x axis. MV-specific IgG titers were measured by ELISA on days 0, 28, and 56. Bars show GM titers (EU/ml) ± the SD.

FIG. 5.

Serum PRN titers from mice i.m. immunized with DNA vaccines encoding MV-H and/or MV-F. Sera from the experiment outlined in Fig. 4 were assessed for their ability to neutralize MV replication in Vero cells using the plaque reduction neutralization assay. PRN titers were measured using sera prepared on days 0 and 56. Responses are expressed as GM titers (mIU/ml) ± SD.

FIG. 6.

Avidity of MV-specific serum IgG in mice immunized i.m. with DNA vaccines encoding MV-H and/or MV-F. Sera from day 56 of the experiment outlined in Fig. 4 were assessed for avidity. Bars show the percent binding after urea treatment ± SD from replicate measurements.

FIG. 7.

Measles virus-specific IFN-γ responses. On day 70, mice were sacrificed, their spleens were extracted and homogenized, and splenocytes were isolated. Single-cell suspensions were pooled within each group and restimulated in vitro for 48 h with 5 μg/ml MV lysate in an IFN-γ-specific ELISPOT assay. Results are expressed as the mean IFN-γ spot-forming cells (SFC) ± SD from triplicate wells/10⁶ cells. IFN-γ spots in wells containing medium (controls) were subtracted from the spots counted in wells containing MV for all experimental groups.