Barbara E.H. Coupar

Vaccinia infection interferes with the presentation of influenza Haemagglutinin (HA) and Nucleoprotein (NP) to class I-restricted CTL. The inhibitory effect is selective for certain epitopes, and is more profound during the late phase of infection. For influenza A/NT/60/68 NP, the block is present during both early and late phases of infection, and is selective for the COOH-terminal epitope defined by peptide 366-379, having no detectable effect on the presentation of the NH2-terminal epitope 50-63. The presentation of HA is inhibited only during the late phase of vaccinia infection. For both proteins, presentation is partially (NP) or completely (HA) restored by expression of rapidly degraded protein fragments in the vaccinia infected target cell. For HA, deletion of the NH2-terminal signal sequence completely overcomes the block. For NP, either a large NH2-terminal deletion or the construction of a rapidly degraded ubiquitin-NP fusion protein partially restores presentation. These results illustrate the relationship between degradation of viral proteins in the cytoplasm of an infected cell and recognition of epitopes at the cell surface by class I-restricted T cells.

Regulation of the expression of influenza A/PR/8/34 hemagglutinin (HA) by the vaccinia virus promoters PF (early), P7.5 (early and late) and PL11 (late) has been demonstrated using HA-vaccinia recombinant viruses VV-PR8-HA3, VV-PR8-HA6 and VV-PR8-HA, respectively. Levels of HA on the surface of VV-PR8-HA3 (PF)-infected cells were lower than with either VV-PR8-HA6 (P7.5) or VV-PR8-HA8 (PL11). Expression of HA under the control of the late promoter PL11 was inhibited in the absence of DNA replication. All three recombinant viruses stimulated a specific antibody response in mice which was dependent on the presence of infectious virus. Recognition of HA by cytotoxic T lymphocytes (CTL) was assessed by the ability of the viruses to stimulate naive precursors in vivo, to restimulate primed CTL in vitro and by target cell recognition. HA expressed under the control of either of the promoters with early function (PF or P7.5) was recognized by CTL when VV-PR8-HA3 or VV-PR8-HA6 were used to prime or restimulate splenocytes or to infect target cells. On the other hand, HA expressed by VV-PR8-HA8 (PL11) failed to prime for a CTL response in naive CBA/H mice, was ineffective at restimulation of primed splenocytes and failed to produce target cells for recognition by specific CTL. However, in BALB/c mice VV-PR8-HA8 did prime for a specific CTL response. These studies show that HA synthesized early in infection was recognized by both B and T cells while HA expressed after DNA replication was not generally recognized by T cells. The implications of the observations with the late promoter with respect to the use of late promoters in potential vaccinia virus-based vaccines are considered.