Deletion of the A35 gene from Modified Vaccinia Virus Ankara increases immunogenicity and isotype switching

Abstract

We show here that the immunogenicity of the Modified Vaccinia Ankara MVA vaccine strain can be improved by deletion of the A35 gene, without diminishing the ability of the virus to replicate. Deletion of the A35 gene resulted in increased virus-specific immunoglobulin production, class switching to IgG isotypes, and virus-specific IFNγ-secreting splenocytes. The MVA35 deletion virus provided excellent protective efficacy against virulent virus challenge. These results suggest that A35 deletion mutant strains will have superior vaccine performance for poxvirus vaccines as well as platform vaccines for other infectious diseases and cancer.

Figure 1. Molecular characterization of MVA35Δ

a) PCR. MVA-infected cells were transfected with a recombinant PCR fragment containing the E. coli gpt gene inserted between the A35 flanking regions and recombinant viruses were selected in mycophenolic acid-containing media. Virus crude stocks were PCR analyzed using primers in the A35 flanking regions. Wild type A35 locus yields a product of 1400 kbp size and the mutants with gpt inserted yield a size of approx 1900 kbp. L=ladder, b) Western blot showing that A35 is not expressed in MVA35Δ-infected cells. BHK-21 cells were infected with listed viruses at an MOI of 20 for 2 h and analyzed by SDS-PAGE. Blots were incubated with rabbit anti-A35 antibody at a 1:1000 dilution and developed with an anti-rabbit alkaline phosphatase-conjugated secondary antibody.

Figure 2. A35 is not required for replication of MVA

a) MVA and MVA35Δ mutant viruses form similarly sized foci on BHK-21 monolayers. Virus-infected cells were visualized by immunostaining. b) One step growth curve. BHK-21 cells or CEF were infected (MOI=10) with MVA, MVA35Δ–1, or MVA35Δ-2, and the amount of virus associated with cells and in the supernatants was titered on BHK-21 cells at various times post infection.

Figure 3. Infection of mice with MVA and MVA35Δ

Groups of mice (n=5) were infected i.m. with 10⁷ pfu/mouse of MVA or MVA35Δ virus, or mock-vaccinated with PBS and weighed (g +SD) for 4 weeks. No mice lost weight or showed signs of illness.

Figure 4. VACV-specific antibody response

Mice (PBS n=3 day 7, n=4 day 10; MVA35Δ n=4; MVA n=4) were infected i.m. with MVA or MVA35Δ virus, and blood was collected via cardiac stick on various days pi. Sera were titrated 1:2, dilutions ranging from 1:20–1:2560. Total VACV-Ig was measured by ELISA on VACV coated plates for day 7 and 10. Data show the average absorbance (day 7 – 1:80, day 10 – 1:20) at 405 nm (+/− SEM).

Figure 5. VACV-specific antibody isotype response

Mice (n=3–5) were infected i.m. with MVA or MVA35Δ virus, and blood was collected on various days pi. Sera were diluted 1:2, resulting in dilutions ranging from 1:10–1:1280. VACV-specific IgM, IgG, IgG1, and IgG2a were measured by ELISA on VACV coated plates for day 7 and 10. Data show the average absorbance (1:20 dilution) at 405 nm (+/− SEM).

Figure 6. VACV-specific IFNγ-producing cells

a) IFNγ production. On days 6 and 10 pi (i.m.), the spleens from 5 mice/group were harvested and splenocytes were analyzed by ELISPOT for virus-specific IFNγ production 48 h after stimulation with VACV-WR virus. b) CD8+ cells. On days 7 and 10 pi, spleens from vaccinated mice were analyzed by flow cytometry for the percentage of CD8+ T cells. Data show the average (+/− SEM).

Figure 7. Cellular subsets in spleens

On day 6 pi, spleens from MVA and MVA35Δ-infected mice (n=5) were stained for various cell surface markers to enumerate percentage of different cell types. Data show average percentage (+/− SEM). Gran, granulocytes; NK, natural killer; MO, macrophage; DC, dendritic cell.

Figure 8. Protection from lethal challenge

Mice (n=5) were vaccinated i.m. on day 0 with 10⁷ pfu of MVA or MVA35Δ virus, or mock-vaccinated with PBS. Four weeks later, the mice were challenged i.n. with LD50 × 500 virulent WR virus. Data show average percent change in pre-challenge weight (+/− SEM). All mice died in the PBS mock vaccinated group.