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 lifelong (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-gamma 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.

Mutations in the temperature‐sensitive murine cytomegalovirus (MCMV) mutants tsm5 and tsm30: A study of genes involved in immune evasion, DNA packaging and processing, and DNA replication

“…Mice inoculated with as little as 4 pfu of tsm5 were protected from a lethal wt virus challenge [Morley et al, 2002]. Tsm5 did replicate, albeit with considerably delayed kinetics, in SCID mice but, unlike with parental virus, which killed all inoculated mice within 16 days, no tsm5-infected mice had died by 28 days post-inoculation [Morley et al, 2002].…”

“…Tsm5 expressed all kinetic classes of transcript [immediate-early (IE), early (E), and late (L)] both in vitro at the nonpermissive temperature and in vivo during acute infection; furthermore, following immunosuppression in vivo IE, E and L genes were expressed without production of infectious virus [Bevan et al, 1996;Gill et al, 2000]. Mice inoculated with as little as 4 pfu of tsm5 were protected from a lethal wt virus challenge [Morley et al, 2002]. Tsm5 did replicate, albeit with considerably delayed kinetics, in SCID mice but, unlike with parental virus, which killed all inoculated mice within 16 days, no tsm5-infected mice had died by 28 days post-inoculation [Morley et al, 2002].…”

Use of a Murine Cytomegalovirus K181-Derived Bacterial Artificial Chromosome as a Vaccine Vector for Immunocontraception

“…The K181 strain of MCMV has also been used in vaccination studies (35), and we have used the K181 Perth strain of MCMV as a vaccine vector (24). Consequently, we set out to develop a K181 BAC that would facilitate all components of our research.…”

“…However, for use in humans, it may be important to produce vaccine vectors that are attenuated because of the number of immunocompromised individuals in the population. This should be technically feasible, as vaccine strains of MCMV that replicate poorly in vivo yet protect from challenge with virulent wild-type (wt) MCMV have been produced (26,35). All these data suggest that CMV may make a useful, safe vaccine vector that can induce long-lived humoral and cell-mediated immune responses to heterologous antigen.…”

“…We and others have used the K181 strain of MCMV extensively for studies on MCMV host-virus interactions (5,10,35,45,49,53). The K181 strain of MCMV has also been used in vaccination studies (35), and we have used the K181 Perth strain of MCMV as a vaccine vector (24).…”