Recombination-Mediated Genetic Engineering of a Bacterial Artificial Chromosome Clone of Modified Vaccinia Virus Ankara (MVA) - Genetic Engineering: Recent Developments in Applications - page 51

exogenous protective antigens or candidate 'molecular adjuvants' with the poten-
tial to increase vector immunogenicity. The elegance of the GalK based recom-
bineering system [55] relies on the ability to counterselect for clones lacking GalK
using deoxygalactose, which is metabolised to a toxic intermediate. This enables
insertion of genes without the need for a selectable marker. However, loss of GalK
can occur both by recombination with an electroporated DNA, or by deletion
of a segment of the BAC. Unlike GalK insertion, which has an efficiency close
to 100% in MVA-BAC, the specific replacement of GalK with an exogenous
sequence has been achieved to date at a frequency of only up to 1% compared to
spontaneous deletions.
We used this technique to insert an antigen expression cassette carrying no
tandem reporter gene or selectable marker into the traditional thymidine kinase
(TK) locus of MVA-BAC. This construct comprised the p7.5 early/late promoter
driving an 82 amino acid epitope string, named “TIP” (for Tuberculosis, Im-
munodeficiency virus and Plasmodium), which contains relevant epitopes from
various pathogens for vaccine-induced protection in mouse models, including
the protective H-2Kd-restricted Pb9 epitope from Plasmodium berghei circum-
sporozoite protein [67]. Figure 5 shows that Pb9-specific CD8+ T cell responses
are elicited in MVA-BAC-TIP immunised mice to a level comparable with those
elicited by conventional MVA-TIP. Although not statistically significant, there is
a trend toward higher Pb9 responses (p = 0.07, t-test) and lower E3 responses (p
= 0.11, t-test) in the conventionally derived MVA-TIP compared to the BAC-
derived virus. This difference in immunodominance hierarchy may be attribut-
able to differences in the nature of the cassette inserted at the TK locus (the lack
of a late-promoter-driven GFP marker gene and opposite orientation of the TIP
gene) or to the presence of the BAC cassette at deletion III.
Figure 5. Immunogenicity of MVA-BAC expressing a recombinant antigen inserted at the standard
thymidine kinase insertion site, in comparison to conventional recombinant MVA and recombineering
precursors containing no insertion or GalK. The recombinant antigen, TIP, is an epitope string containing
the Pb9 epitope from Plasmodium berghei circumsporozoite protein [67]. Bars show mean specific CD8 + T cell
responses to the indicated peptides 7 days after i.d. vaccination of with 10 6 pfu, from groups of four BALB/c
mice, with error bars showing SEM.