Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
Correspondence
Philip Stevenson
pgs27{at}mole.bio.cam.ac.uk
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ABSTRACT |
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MAIN TEXT |
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It is possible with the murine gammaherpesvirus-68 (MHV-68) to test the capacity of different vaccination strategies to protect against a gammaherpesvirus infecting what is probably its natural host (Blasdell et al., 2003). Subunit vaccines including gp150, the MHV-68 homologue of the EBV gp340 (Stewart et al., 1996
) have so far proved ineffective in controlling MHV-68 latency establishment (Stewart et al., 1999
; Stevenson et al., 1999a
; Liu et al., 1999
; Usherwood et al., 2001
). The multiple immune evasion strategies of MHV-68 (Stevenson et al., 2002a
) probably render ineffective the limited immunity that is generated against isolated viral components. Since an empirically attenuated strain of varicella-zoster virus (Asano & Takahashi, 1977
) is the only herpesvirus vaccine to date that has proved clearly effective in preventing disease in the natural host (Wills et al., 2002
), attenuated MHV-68 mutants may elicit more effective immune protection than individual viral subunits. The existence of multiple co-existing EBV strains in some individuals (Brooks et al., 2000
; Sitki-Green et al., 2003
, Walling et al., 2003
) suggests that sterilizing immunity to gammaherpesvirus infections is probably not a realistic goal. However, it may be possible to reduce the long-term level of latency and so prevent disease.
Because it is possible with MHV-68 to identify the contribution of individual gammaherpesvirus genetic loci to pathogenesis and host colonization, we can improve on empirical strategies of attenuation and employ specific, testable, molecular-based strategies to design candidate vaccines. In the accompanying paper (May et al., 2004) we have described the generation and characterization of a latency-deficient mutant of MHV-68, M50, made by deregulating the transcription of the viral ORF50 lytic transactivator. We targeted viral latency because this makes a major contribution to MHV-68 host colonization (Coleman et al., 2003
) and because latency-associated lymphocyte proliferation probably underlies most clinically important, gammaherpesvirus-associated disease. A major immediateearly transactivator that initiates the viral lytic cycle is common to all gammaherpesviruses, even though the transactivators themselves are relatively varied (Sun et al., 1998
; Wu et al., 2000
; Binne et al., 2002
). The MHV-68 ORF50 is homologous to the ORF50 of other gamma-2-herpesviruses, including the Kaposi's sarcoma-associated herpesvirus (KSHV) and to the Rta transactivator of EBV. Thus the strategy of MHV-68 attenuation by deregulation of ORF50 could be readily adapted to other gammaherpesviruses that cause clinically and economically important disease. Here we have tested the capacity of infection with the M50 mutant to protect against host colonization following a subsequent WT virus challenge. The aim was to provide a proof of principle that the immunity to a latency-deficient mutant can protect against the subsequent establishment of normal latency by WT virus.
We first measured the immune response to M50 infection (Fig. 1). The virus-specific CD4+ T cell (ELISpots with virus-infected targets), CD8+ T cell (ELISpots with epitope-pulsed targets) and B cell (serum antibody) responses were all reduced compared to those elicited by infection with WT or revertant (50R) viruses. The IFN-
-producing CD4+ T cell response was the most obviously affected, while the differences in virus-specific serum antibody titres were relatively small. The reduced immunogenicity of the M50 virus presumably reflected both its reduced lytic replication in the lung and its failure to achieve latency amplification (see May et al., 2004
). Lytic recrudescence from the pool of genome-positive cells generated by latency amplification provides a surprisingly large load of immunogenic MHV-68 lytic antigens (Stevenson et al., 1999c
, 2002b
; Coleman et al., 2003
), so we would expect most latency-deficient viruses to be less immunogenic than WT. However, despite its reduced immunogenicity, the M50 virus still offered significant theoretical advantages over subunit vaccines, due to the range of antigens presented and the appropriate context of their presentation. We chose to retain the M3 and MK3 immune evasion genes in the M50 vaccine virus again to provide appropriate levels of antigen in an appropriate infectious context. MK3-deficient MHV-68 elicits higher frequencies of virus-specific CD8+ T cells than WT virus (Stevenson et al., 2002b
), but the capacity of these cells to control a virus with intact immune evasion remains unproven; CD8+ T cells stimulated by high levels of MHC class I/peptide complexes are not always effective when the numbers of these complexes on infected target cells are small.
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To test the protection afforded by M50-specific immunity, we vaccinated mice intranasally with M50 virus (2x104 p.f.u.) and 3 months later infected these mice and age-matched, unvaccinated controls intranasally with WT virus (2x104 p.f.u.). There was an almost complete ablation of lytic virus replication in the lungs and latent viral amplification in the spleens of the vaccinated mice (Fig. 2). There was also no evidence in the vaccinated mice of the latency-associated splenomegaly or CD8+V
4+ T cell expansion normally driven by WT virus. Thus even the limited immunity afforded by M50 infection was sufficient to control WT viral lytic replication and latency amplification.
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Disease prevention relevant to EBV or KSHV is difficult to assess directly in the MHV-68 model because the murine virus, despite one report (Sunil-Chandra et al., 1994), is not obviously tumorigenic in standard mouse strains, even with immune suppression. Thus a direct correlate of the desirable outcome of an EBV or KSHV vaccine preventing tumours is currently not possible. However, despite not being tumorigenic, MHV-68 does drive lymphocyte proliferation. This results in a massive increase in latently infected B cell numbers in the infectious-mononucleosis-like illness, with widespread lymphocyte activation and a marked expansion in the CD8+V
4+ T cell subset (Doherty et al., 1997
). Thus a reduction in MHV-68 latency establishment and MHV-68-driven mononucleosis probably reflects a reduction in virus-driven lymphocyte proliferation, the process that underlies gammaherpesvirus-associated tumours. M50-vaccinated mice did show a clear inhibition of latency establishment by superinfecting WT virus.
The fact that the M50 virus itself is impaired in latency amplification meant that the total latent viral load in vaccinated mice remained low. It is possible that M50 virus might accumulate compensatory mutations to prevent entry into the lytic cycle and so maintain latency. However, the primed immune response should limit the size of an M50 latent pool in the same way that it limited the WT latent load. It seems unlikely that vaccination with the M50 virus can completely prevent WT infection each PCR reaction, after all, samples only a small fraction of the whole organ but it did appear possible to limit WT virus-driven lymphocyte proliferation without this being a characteristic of the immunizing virus. It should presumably be possible to employ a similar strategy with other gammaherpesviruses so as to protect against the disease associated with WT viral latency.
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ACKNOWLEDGEMENTS |
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Received 21 August 2003;
accepted 10 October 2003.