Recombination-Mediated Genetic Engineering of a Bacterial Artificial Chromosome Clone of Modified Vaccinia Virus Ankara (MVA) - Genetic Engineering: Recent Developments in Applications - page 49

Immunogenicity of MVA-BAC Deletion Mutants
The effect of the above modifications on immunogenicity was assessed in intra-
dermally immunised BALB/c mice. Peak specific T cell responses were analysed
seven days later by flow cytometry with intracellular cytokine staining following
stimulation with the F2(G) and E3 peptides (Figure 3). No statistically significant
differences between groups were observed (one-way ANoVA for each epitope). Of
the selected genes, only B15R, which encodes an IL-1 β -binding protein, has been
reported to affect MVA immunogenicity, rather than Vaccinia virus immunoge-
nicity or virulence [51]. At the peak of the response, Staib et al. described a small
non-significant effect of B15R deletion, very similar to that shown in Figure 3 for
the E3 epitope, and with an identical p-value by t-test (p = 0.07); however, in the
memory phase six months post-vaccination, they observed a highly significant
~4.5-fold increase in CD8+ T cell response to the B15R deletion mutant. We
therefore assessed memory responses at 8 weeks post-vaccination, and observed
a small, but statistically significant increase in CD8+ T cell responses to E3 (p =
0.039, t-test), but not to F2(G). As well as the readout time, variation in dose
(108 vs. 106 pfu), route (i.p. vs. i.d.), antigen (H3 vs. E3 peptide) and mouse
strain (HHD vs. BALB/c) are likely to underlie the suggested difference in the
magnitude of the effect of B15R deletion on memory T cell responses.
Figure 3. Peptide-specific splenic CD8+ T cell responses 7 days (d7) or 56 days (d56) after i.d. immunisation of
BALB/c mice with 106 pfu of MVA-BAC derived viruses, either unmodified or with indicated genes deleted by
insertion of GalK.* p = 0.039 (t-test). Data are means and SEM from groups of four mice.
In order to characterise the effect of B15R deletion further, we analysed E3-
specific CD8+ T cell expression of TNF- α and IL-2 in addition to IFN- γ . SPICE
software [66] was used to discriminate individual cells expressing the seven dif-
ferent 'Boolean' combinations of cytokines from the IFN- γ +, TNF- α + and IL-2+
gated populations (Figure 4). This approach reveals that at the peak of the response