DNA immunization against herpes simplex virus: enhanced efficacy using a Sindbis virus-based vector

Abstract

Previously we reported the development of a plasmid DNA expression vector system derived from Sindbis virus (T. W. Dubensky, Jr., et al., J. Virol. 70:508-519, 1996). In vitro, such vectors exhibit high-level heterologous gene expression via self-amplifying cytoplasmic RNA replication. In the present study, we demonstrated the in vivo efficacy of the Sindbis virus-based pSIN vectors as DNA vaccines. A single intramuscular immunization of BALB/c mice with pSIN vectors expressing the glycoprotein B of herpes simplex virus type 1 induced a broad spectrum of immune responses, including virus-specific antibodies, cytotoxic T cells, and protection from lethal virus challenge in two different murine models. In addition, dosing studies demonstrated that the pSIN vectors were superior to a conventional plasmid DNA vector in the induction of all immune parameters tested. In general, 100- to 1,000-fold-lower doses of pSIN were needed to induce the same level of responsiveness as that achieved with the conventional plasmid DNA vector. In some instances, significant immune responses were induced with a single dose of pSIN as low as 10 ng/mouse. These results indicate the potential usefulness of alphavirus-based vectors for DNA immunization in general and more specifically as a herpes simplex virus vaccine.

FIG. 1

Schematic diagram of plasmid DNA-based expression vectors used for DNA immunization with HSV-1 gB (diagram is not drawn to scale). Individual elements comprising the functional expression cassettes for both conventional (pCI-gB) and Sindbis virus (pSIN1.5-gB and pSIN2.5-gB) plasmid vectors are indicated. Sindbis virus-derived sequences are shown in white and include the four nonstructural protein genes (nsPs), complete 5′- and 3′-end untranslated regions, subgenomic promoter (JR), and poly(A) tract (A₄₀). Shaded regions depict the CMV immediate-early promoter (CMV) and intron (INT), hepatitis delta virus antigenomic ribozyme sequence (δ), simian virus 40 late-region transcription termination signal (TT) in the pCI vector, bovine growth hormone transcription termination signal (TT) in the pSIN vectors, hepatitis B virus posttranscriptional regulatory element (PRE), and HSV-1 gB.

FIG. 2

Expression of HSV-1 gB in vitro from pCI and pSIN vectors. Cell lysates were prepared from BHK-21 cells transfected with the different plasmid vectors. Cell lysates were harvested 48 h posttransfection, separated on SDS–8 to 16% polyacrylamide gels, transferred, and probed with a mouse monoclonal anti-HSV-1 gB antibody followed by a goat anti-mouse IgG2a-HRP antibody. Sizes are indicated in kilodaltons.

FIG. 3

Induction of HSV-1-specific total IgG antibody in mice immunized with pCI or pSIN vectors expressing gB. BALB/c mice given a single i.m. injection of the different vectors at either 3.0 or 0.3 μg/mouse were bled at day 14 postimmunization, immediately prior to lethal challenge with HSV-1 McKrae. ELISA was used to measure total IgG antibody titers in individual mice. The normal serum control represents a pool from several nonimmune mice.

FIG. 4

Induction of HSV-1-specific total IgG antibody in mice immunized with pCI or pSIN vectors expressing gB. BALB/c mice given a single i.m. injection of the different vectors at 1.0, 0.3, or 0.05 μg/mouse were bled at 15 weeks postimmunization, and ELISA was used to measure total IgG antibody titers in individual mice. The normal serum control represents a pool from several nonimmune mice.

FIG. 5

Induction of HSV-1 gB-specific CTL in mice immunized with pCI or pSIN vectors expressing gB. Splenocytes from individual mice collected after a single i.m. injection with the pCI-gB or pSIN-gB vectors were restimulated in vitro with BC-gB1 and tested for cytolytic activity in a 4-h ⁵¹Cr release assay using BC-gB1 target cells (closed symbols) and BC-βgal target cells (open symbols). Cytolytic activity is shown as the percent specific lysis detected at each effector/target cell ratio tested and represents the mean of triplicates. This experiment was repeated on two additional occasions with similar results.