Recombination-Mediated Genetic Engineering of a Bacterial Artificial Chromosome Clone of Modified Vaccinia Virus Ankara (MVA) - Genetic Engineering: Recent Developments in Applications - page 50

(d7), most of the cells are IFN- γ and TNF- α double-positives. Furthermore, al-
though there is no conventionally significant difference, the increase in bulk
IFN- γ responses observed with the B15R deletion mutant is primarily the result
of an increased number of these double-positive cells, rather than of the IFN- γ
single-positive or the IFN- γ , TNF- α and IL-2 triple-positive cells that account for
the remainder of the CD8+ T cell response. By contrast, at day 56, the effect of
B15R deletion is seen to reside mainly within the triple-positive population, and
the difference is statistically significant (p = 0.018, Willcoxon-Rank test). Dele-
tion of B15R therefore affects both the quality and magnitude of the E3-specific
CD8+ T cell response to MVA. Enhanced production of the mitogenic cytokine
IL-2 during the early memory phase (8 weeks) may underpin the large difference
in T cell frequency observed in Staib et al.'s 6 month experiment [51].
Figure 4. Multifunctionality of CD8 + T cells induced 7 days (d7) or 56 days (d56) after immunisation of
BALB/c mice with unmodified MVA-BAC or B15R deletion mutant. Histograms show frequency of cells
expressing each of the seven possible combinations of IFN- γ , IL-2 and TNF- α with bars showing the mean and
circles showing the values for individual mice (n = 4). Pie charts show fraction of the total response comprised
of cells expressing all three cytokines (black), any two cytokines (dark gray), or one cytokine only (light gray). *
p = 0.018, Willcoxon-Rank test. Analysis performed using SPICE software [66].
Insertion of a Recombinant Antigen into MVA by BAC
recombineering
In addition to more rapid production of deletion mutants, the MVA-BAC sys-
tem would also have value as a faster method for insertion of genes encoding