Immunodominant vector-specific CTL responses could limit their
effectiveness especially in cancer immunotherapeutic strategies
implying multiple rounds of vaccine stimulations. We aim at
decreasing CTL responses against Vaccinia Virus by diminishing the
viral epitope MHC class-I restricted presentation from infected cells.
ICP47 protein (encoded by US12 gene from HSV-I) is inhibiting TAPdependant
transport to the endoplasmic reticulum (ER) of processed
peptide thus preventing their class-I presentation. We hypothesized
that co-expression of HSV-ICP47 in a recombinant vaccinia virus
(rVV) would prevent presentation of viral proteins without affecting the
presentation of recombinant TAP-independent tumor associated
antigen (TAA) epitopes. MHC class-II presentation of viral entities,
which play a major role as "helper signal" during the generation of
CD8+ response, should not be affected.
Methods: Herpesvirus US12 gene was inserted into wild type
genome as well as into a rVV expressing ER-Mart27-35. Replication
of the virus is prevented by psoralen-UV treatment. Effect on MHCclass
I of infected cells was characterized by antibody staining and
FACS analysis.
Human T-lymphocytes were stimulated in vitro with autologous
CD14+ cells infected with US12-rVV, M-US12- rVV or control virus.
Proliferation of specific CD8+ and CD4+ for viral proteins and the
recombinant epitope were monitored by MHC-multimer and IFNg
intracellular staining.
Results: Already 16-24h after infection, cells transfected with US12-
rVV demonstrated a clear MHC class-I downregulation. In HLA-A0201
positive cell lines, downregulation of the HLA-A2 complex observed
with US12-rVV was partially compensated by the co-expression of
ER-Mart27-35 peptide encoded in M-US12-rVV. No effect of
US12-rVV was seen for other surface molecules such as CD44,
CD80 and MHC class-II, confirming the specificity of the blockade.
Finally, preliminary tests seem to indicate that CD8+ responses
against viral epitopes (processed from vaccinia vector) are diminished
when primed with US12-rVV.
Conclusion: Recombinant vaccinia virus expressing the HSV-US12
gene confirmed a diminished class-I presentation of native viral
proteins from infected cells. Whereas the immunogenicity of
recombinant ER-targeted class-I TAA epitope, as well as viral helper
class-II entities, should be conserved. Such reagent could become of
high relevance especially in multiple-boost vaccine protocol required
in cancer immunotherapy.