DNA immunization with a herpes simplex virus 2 bacterial artificial chromosome

Abstract

Construction of a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) is described. BAC vector sequences were inserted into the thymidine kinase gene of HSV-2 by homologous recombination. DNA from cells infected with the resulting recombinant virus was transformed into E. coli, and colonies containing the HSV-2 BAC (HSV2-BAC) were isolated and analyzed for the expected genotype. HSV2-BAC DNA was infectious when transfected back into mammalian cells and the resulting virus was thymidine kinase negative. When used to immunize mice, the HSV2-BAC DNA elicited a strong HSV-2 specific antibody response that was equal to or greater than live virus immunization. Further, HSV2-BAC immunization was protective when animals were challenged with a lethal dose of virus. The utility of the HSV2-BAC for construction of recombinant virus genomes was demonstrated by elimination of the HSV-2 glycoprotein D (gD) gene. A recombinant HSV-2 BAC with the gD gene deleted was isolated and shown to be incapable of producing infectious virus following transfection unless an HSV gD gene was expressed in a complementing cell line. Immunization of mice with the HSV2 gD-BAC also elicited an HSV-2 specific antibody response and was protective. The results demonstrate the feasibility of DNA immunization with HSV-2 bacterial artificial chromosomes for replicating and nonreplicating candidate HSV-2 vaccines, as well as the utility of BAC technology for construction and maintenance of novel HSV-2 vaccines. The results further suggest that such technology will be a powerful tool for dissecting the immune response to HSV-2.

Fig. 1

Schematic diagram of the structure of HSV2-BAC. The BeloBAC vector sequences were inserted into the HSV-2 thymidine kinase gene via homologous recombination between HSV-2 and the insertion vector pSP72tkBeloBAC. The single XhoI restriction site in BeloBAC is approximately 2 kb from the HindIII site used for insertion into the tk gene of pSP72tkBeloBAC.

Fig. 2

Southern blot analysis of HSV2-BAC. The DNA from two independently isolated bacterial clones containing the HSV2-BAC DNA was analyzed by XhoI restriction digest and Southern blotting. HSV-2 DNA (lane 2) and HSV2-BAC DNA (lanes 1 and 3) were hybridized with a 3.47-kb (coordinates 45 174–51 311) thymidine kinase sequence probe (A) or a BeloBAC vector probe (B) to confirm the correct site of insertion of BeloBAC sequences. The size of DNA markers (M) are indicated in kilobase pairs to the left of each figure.

Fig. 3

Virus replication analysis of HSV-2 and HSV2-BAC virus. Vero cells (5 × 10⁵) were infected with HSV-2 or HSV2-BAC virus at an moi of 0.1 in the presence or absence of acyclovir. Total virus yields at the indicated times postinfection were determined by plaque assay on Vero cells.

Fig. 4

Antibody response following immunization with HSV2-BAC DNA. Mice were immunized two times with 1 μg of HSV2-BAC DNA, 5 × 10⁴ pfu of HSV2-BAC virus, 5 × 10² pfu of HSV2-BAC virus, or 1 μg of pBeloBAC control DNA. Results from a representative experiment, of two experiments of similar design and outcome, are shown. Each group consisted of five animals. (A) HSV-2 specific IgG was determined for individual mice 3 weeks following the first immunization and 3 weeks following a booster immunization with the same dose of either DNA or virus. The number of mice with a detectable IgG titer (≥1:20) is indicated if less than five. Calculation of the mean IgG titer includes a value of 1:10 for sero-negative animals. (B) The titer of HSV-2 specific IgG isotypes was determined 3 weeks after 2 immunizations with 1 μg of HSV2-BAC DNA (DNA) and 5 × 10⁴ pfu of HSV2-BAC virus (virus). The number of mice with a detectable antibody titer (≥1:40) is indicated if less than five. Calculation of the mean IgG titer includes a value of 1:20 for sero-negative animals.

Fig. 5

Lethal challenge of HSV2-BAC DNA- and HSV2-BAC virus-immunized mice. Three weeks following the booster immunization with HSV2-BAC DNA, HSV2-BAC virus, or control DNA, mice were challenged intraperitoneally with 5 × 10⁶ pfu (approximately 25 LD₅₀) of HSV-2 (MS). Survival curves are shown for each group of animals.

Fig. 6

Antibody response following immunization with HSV2-BAC and HSV2 gD-BAC DNA. Mice were immunized with 1 μg of pBeloBAC control DNA, HSV2-BAC DNA, or HSV2 gD-BAC DNA. Serum samples were collected and analyzed for HSV-2 specific IgG at 3 weeks after the first immunization and 3 weeks following a booster immunization of the same dose of DNA at 3 weeks. Results from a representative experiment, of 2 experiments of similar design and outcome, are shown.

Fig. 7

Lethal challenge of HSV2-BAC DNA- and HSV2 gD-BAC DNA-immunized mice. Three weeks following the booster immunization with HSV2-BAC DNA, HSV2 gD-BAC DNA, or control DNA, mice were challenged intraperitoneally with 2 × 10⁶ pfu (approximately 10 LD₅₀) of HSV-2 (MS). Survival curves are shown for each group of animals.