Biology Reference
In-Depth Information
discussion
Traditional methods for genetic manipulation of poxviruses, either for mutagen-
esis or expression of exogenous genes, rely on onerous plaque purification of rare
recombinant viruses. Building on the results of Domi and Moss for VAC-BAC
[53], [54], here we circumvent this requirement by creating an MVA-BAC that
can be manipulated using recombineering and rescued to clonal viruses. With an
efficiency similar to that observed in the case of VAC-BAC, we generated four
MVA-BAC clones, which were identical by restriction map and had indistin-
guishable CD8
+
T cell immunogenicity in two strains of mice. We sequenced one
clone, and found it to be identical to published genomic sequences of MVA, with
the exception of a minor mutation that is polymorphic amongst Vaccinia virus
strains and was present in the parent MVA recombinant. We did not conduct an
investigation into stability of the construct, since good stability had been shown
for the even larger VAC-BAC construct [53], [54].
Of five candidate genes selected from the literature for deletion by MVA-
BAC recombineering (Table 2), only one had any statistically significant effect
on responses to viral CD8+ T cell epitopes following intradermal immunisation
of BALB/c mice. The effect of B15R deletion was more pronounced 8 weeks
post-vaccination, though not as great as was previously reported at a 6 month
timepoint in HHD mice [51]. Although we did not observe an effect of similar
magnitude using an earlier readout, we show using SPICE polychromatic flow
cytometry analysis that antigen-specific CD8+ T cells from mice immunised with
MVA lacking B15R are composed of a higher proportion of triple-positive cells
that express IL-2 in addition to IFN-
γ
and TNF-
α
. This 'multi-functional' phe-
notype may be responsible for the large differences in cell frequency at 6-months
post-vaccination [51], since IL-2 is strongly implicated in memory T cell homeo-
stasis [68] and its expression by virus-specific CD8+ T cells has been reported to
promote antigen-specific proliferation of these cells even in the absence of CD4+
T cell help [69]. Whether responses to a recombinant antigen will be augmented
in a B15R deletion background remains to be tested, though it should be noted
in this regard that enhancement of anti-vector responses was confined to the E3-
specific response and was not observed in the case of the F2(G) epitope.
Deletion of four other genes by GalK insertion in MVA-BAC did not po-
tentiate CD8+ T cell responses to the vector epitopes. C12L, A44R and B7R all
carry MVA-specific mutations that do not occur in any other sequenced strain of
Vaccinia virus. Although strain Ankara (from which MVA was derived) has not
been sequenced, these mutations presumably arose during attenuation, and the
genes affected may therefore already be inactive. Alternatively, or additionally,
functional redundancy of families of immunomodulatory genes acting on the
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