Immunisation of Balb/c mice with severely attenuated murine cytomegalovirus mutants induces protective cellular and humoral immunity

Abstract

Previously, we showed that two temperature-sensitive mutants of murine cytomegalovirus (tsm5 and tsm30) expressed immediate-early (IE-1), early (E-1), and late (gB) phase genes in the tissues of immunocompetent Balb/c mice, yet failed to produce infectious progeny virus in any tissue at any time at 1-21 days post-infection. Mice inoculated intraperitoneally with tsm5 became latently infected, but this latent virus could not be reactivated as an infectious virus after immunosuppression, although all three transcripts were produced. Immunocompetent mice infected with tsm30 did not become latently infected. In the present study, immunodeficient SCID mice supported productive infection of both mutants, suggesting that low-level viral replication does occur in immunocompetent mice, but that it is limited by the host immune response. This is supported by the observation that immunocompetent mice were protected against virulent K181 challenge even after immunisation with as few as 40 pfu of mutant virus, whereas UV-inactivated mutant or K181 virus was not immunoprotective at doses of 40,000 pfu. Immunity induced by subcutaneous inoculation was also protective, whereas that induced by intragastric immunisation was not. Protection was life-long (18 months). Although tsm5 induced high antibody titres, there was little evidence of an antibody response to tsm30. In contrast, a significant CD8+ CTL response to the Balb/c immunodominant IE-1 nonapeptide (YPHFMPTNL) was elicited by both mutants, as determined by an interferon-γ ELISPOT assay, although this response was lower than that induced by K181 infection. In addition, CTLs specific for m04 (YGPSLYRRF) and M84 (AYAGLFTPL) peptides could be detected at low frequency after K181, tsm5, and tsm30 immunisation. Such protective immunity did not prevent the challenge K181 virus from entering the latent state, but it appeared to reduce the frequency of reactivation. © 2002 Wiley-Liss, Inc.