Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
Correspondence
Stacey Efstathiou
se{at}mole.bio.cam.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
Initial attempts to protect against MHV-68 infection involved priming different arms of the immune response to limit viral infection, for example priming T-cells with defined lytic antigens prior to challenge with MHV-68 (Liu et al., 1999; Stevenson et al., 1999a
, b
). While this was effective in limiting acute infection, it was unable to prevent latency. Similarly, adoptive transfer of CD8+ T cells specific for a latent antigen, M2, and targeted vaccination against this protein reduced the initial load of latently infected cells in the spleen, but had little effect on long-term latency (Usherwood et al., 2000
, 2001
). An important role for the humoral response to MHV-68 infection has also been documented (Sangster et al., 2000
; Stevenson & Doherty, 1998
) and control of MHV-68 infection has been demonstrated by passive transfer of immune serum (Kim et al., 2002
; Tibbetts et al., 2003
). Vaccination using a recombinant vaccinia virus expressing gp150, a major MHV-68 glycoprotein (Stewart et al., 1996
), proved effective in reducing lytic viral titres; however, a latent infection was still established. From these studies it seems that a primed immune response can be effective in limiting the initial load, but has little effect on long-term latency.
Using an alternative strategy, Tibbetts et al. (2003) used an MHV-68 mutant defective in reactivation from latency to immunize against subsequent wild-type (wt) infection. This had the advantage of priming the immunized animal against a range of epitopes and reduced the levels of lytic virus replication during acute infection and long-term latency following wt virus challenge. Importantly, the reactivation-deficient mutant utilized by Tibbetts et al. (2003)
demonstrated that it is possible to reduce long-term latent infection of wt challenge virus using a live attenuated vaccine which itself is capable of establishing latency. In this report we have explored the vaccine potential of an alternative type of virus mutant one that is competent for lytic replication in vivo, but is defective for the establishment and maintenance of long-term latency.
Mutants of MHV-68 deficient for ORF73 are unable to persist in vivo (Moorman et al., 2003; Fowler et al., 2003
). This phenotype is due to the disruption of the latency-associated antigen ORF73 which, by analogy to the homologous ORFs encoded by KSHV and herpesvirus saimiri, is predicted to function in maintenance of the latent viral genome (Ballestas et al., 1999
; Collins et al., 2002
). For herpesvirus saimiri, ORF73 has also been shown to play a role in immediate early gene regulation (Schäfer et al., 2003
). An ORF73-deficient MHV-68 could therefore provide a suitable live attenuated virus that could be utilized as a vaccine as it is competent for lytic replication but defective for latency. The aim of this study was to investigate the ability of an ORF73-defective virus (
73) to confer in vivo protection to challenge with wt MHV-68.
The 73 virus was constructed as described by Fowler et al. (2003)
. Briefly, 450 bp (genomic co-ordinates, 104 379104 830; Virgin et al., 1997
) was deleted from ORF73 within the BamHI G genomic clone (Efstathiou et al., 1990
) and the recombinant virus was made by recombination of the mutated BamHI G fragment and MHV-68 BAC pHA3 (Adler et al., 2000
). The vaccination protocol utilized is depicted in Fig. 1
(a). Briefly, 5- to 6-week-old female BALB/C mice were infected by the intraperitoneal (i.p.) route with 105 p.f.u.
73 virus in PBS/1 % fetal calf serum (FCS) or mock-vaccinated with PBS/1 % FCS. To confirm that the vaccinating virus (
73) had seeded to the spleen, infectious virus was assayed 5 days post-vaccination. Infectious virus was detected in 5/5 animals [mean 2·63±0·63 log10(p.f.u. ml-1)] at levels comparable to that previously observed for an independent ORF73 mutant, FS73, and its revertant virus, FS73R (data not shown) (Fowler et al., 2003
).
|
|
We then determined the effect of vaccination with 73 on the establishment and maintenance of latency following wt virus challenge. We first assessed the latent load in splenocytes from mice vaccinated with
73, 35 days prior to challenge with wt virus. Fourteen days after wt or mock challenge, splenocytes were assayed for infectious centres as a measure of the latent viral load. In mock-vaccinated mice, latency was established to wt levels (Fig. 3
a). In
73-vaccinated, wt-challenged mice no infectious centres were detected (Fig. 3a
). Similarly, no infectious centres were detected in mice that were
73-vaccinated and mock-challenged, consistent with previous observations demonstrating that
73 has a severe latency deficit (Fowler et al., 2003
). Protection against wt virus infection was still evident after 117 days, since infectious centres were not detected in
73-vaccinated mice 14 days post-challenge (Fig. 3b
).
|
|
As the lung has been shown to be another site of MHV-68 latency and persistence (Flano et al., 2003; Stewart et al., 1998
), we also analysed genomic DNA extracted from the lung at 58 days post-challenge to assess the viral load. Real-time PCR was performed on either 100 ng or 1 µg lung DNA. DNA from mock-vaccinated wt-challenged mice was positive for viral DNA in 4/4 mice sampled using both 100 ng and 1 µg template. In contrast, viral DNA could not be detected in 3/3
73-vaccinated mock-challenged or 4/4 wt-virus-challenged mice with either 1 µg or 100 ng lung DNA template, but they were shown to be positive by PCR for a cellular gene, encoding
-actin, confirming the integrity of the DNA template. The PCR assay was sensitive to approximately 10 copies of K3 in 300 ng cellular DNA (data not shown). Our inability to detect viral genomes in
73-vaccinated mice suggests a high level of protection against the establishment of latency following challenge with wt virus.
In this study, we have described the efficacy of vaccination with 73 in protection against acute and latent infection by wt virus. Notably, this protection was observed with a challenge dose 25-fold higher than that described with a reactivation-deficient virus (Tibbetts et al., 2003
). There are several advantages to vaccination using an ORF73-deficient mutant virus. Perhaps the most important of these is that while the
73 virus replicates comparably to wt during acute infection in both the lungs and spleen, therefore presumably priming an effective immune response, this mutant virus is apparently unable to persist in the host (Fowler et al., 2003
). This factor is critical in the context of the gammaherpesviruses due to the association of latent virus genomes and tumorigenesis. The potential transforming ability of gammaherpesviruses indicates that a live virus vaccine that retains its ability to establish latency would have a lower safety profile than an attenuated virus exhibiting a latency deficit. However, while we observed no evidence of latent wt or
73 vaccine virus following vaccination, we cannot conclude that the vaccination is sterilizing. It remains possible that there is a low level of persisting
73 or wt virus that we are unable to detect using the techniques employed in this study.
The aim of this work was to investigate the potential efficacy of a latency-deficient MHV-68 mutant (73) in protection against wt infection. We have shown this vaccination strategy to be highly efficient in limiting acute and latent infection and the protection is long-lived. This study indicates that a live-attenuated gammaherpesvirus virus that cannot persist is an effective vaccine and provides a general basis for the development of safe vaccines against this group of lymphotropic herpesviruses.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
Ballestas, M. E., Chatis, P. A. & Kaye, K. M. (1999). Efficient persistence of extrachromosomal KSHV DNA mediated by latency-associated nuclear antigen. Science 284, 641644.
Collins, C. M., Medveczky, M. M., Lund, T. & Medveczky, P. G. (2002). The terminal repeats and latency-associated nuclear antigen of herpesvirus saimiri are essential for episomal persistence of the viral genome. J Gen Virol 83, 22692278.
Efstathiou, S., Ho, Y. M. & Minson, A. C. (1990). Cloning and molecular characterization of the murine herpesvirus 68 genome. J Gen Virol 71, 13551364.[Abstract]
Flano, E., Kim, I. J., Moore, J., Woodland, D. L. & Blackman, M. A. (2003). Differential gamma-herpesvirus distribution in distinct anatomical locations and cell subsets during persistent infection in mice. J Immunol 170, 38283834.
Fowler, P., Marques, S., Simas, J. P. & Efstathiou, S. (2003). ORF73 of murine herpesvirus-68 is critical for the establishment and maintenance of latency. J Gen Virol 84, 34053416.
Kim, I. J., Flano, E., Woodland, D. L. & Blackman, M. A. (2002). Antibody-mediated control of persistent gamma-herpesvirus infection. J Immunol 168, 39583964.
Liu, L., Usherwood, E. J., Blackman, M. A. & Woodland, D. L. (1999). T-cell vaccination alters the course of murine herpesvirus 68 infection and the establishment of viral latency in mice. J Virol 73, 98499857.
Marques, S., Efstathiou, S., Smith, K. G., Haury, M. & Simas, J. P. (2003). Selective gene expression of latent murine gammaherpesvirus 68 in B lymphocytes. J Virol 77, 73087318.
Moorman, N. J., Willer, D. O. & Speck, S. H. (2003). The gammaherpesvirus 68 latency-associated nuclear antigen homolog is critical for the establishment of splenic latency. J Virol 77, 1029510303.
Sangster, M. Y., Topham, D. J., D'Costa, S., Cardin, R. D., Marion, T. N., Myers, L. K. & Doherty, P. C. (2000). Analysis of the virus-specific and nonspecific B cell response to a persistent B-lymphotropic gammaherpesvirus. J Immunol 164, 18201828.
Schäfer, A., Lengenfelder, D., Grillhösl, C., Wieser, C., Fleckenstein, B. & Ensser, A. (2003). The latency-associated nuclear antigen homolog of herpesvirus saimiri inhibits lytic virus replication. J Virol 77, 59115925.
Stevenson, P. G. & Doherty, P. C. (1998). Kinetic analysis of the specific host response to a murine gammaherpesvirus. J Virol 72, 943949.
Stevenson, P. G., Belz, G. T., Castrucci, M. R., Altman, J. D. & Doherty, P. C. (1999a). A gamma-herpesvirus sneaks through a CD8(+) T cell response primed to a lytic-phase epitope. Proc Natl Acad Sci U S A 96, 92819286.
Stevenson, P. G., Cardin, R. D., Christensen, J. P. & Doherty, P. C. (1999b). Immunological control of a murine gammaherpesvirus independent of CD8+ T cells. J Gen Virol 80, 477483.[Abstract]
Stewart, J. P., Janjua, N. J., Pepper, S. D., Bennion, G., Mackett, M., Allen, T., Nash, A. A. & Arrand, J. R. (1996). Identification and characterization of murine gammaherpesvirus 68 gp150: a virion membrane glycoprotein. J Virol 70, 35283535.[Abstract]
Stewart, J. P., Usherwood, E. J., Ross, A., Dyson, H. & Nash, T. (1998). Lung epithelial cells are a major site of murine gammaherpesvirus persistence. J Exp Med 187, 19411951.
Sunil-Chandra, N. P., Efstathiou, S. & Nash, A. A. (1992). Murine gammaherpesvirus 68 establishes a latent infection in mouse B lymphocytes in vivo. J Gen Virol 73, 32753279.[Abstract]
Tibbetts, S. A., McClellan, J. S., Gangappa, S., Speck, S. H. & Virgin, H. W. (2003). Effective vaccination against long-term gammaherpesvirus latency. J Virol 77, 25222529.
Usherwood, E. J., Roy, D. J., Ward, K., Surman, S. L., Dutia, B. M., Blackman, M. A., Stewart, J. P. & Woodland, D. L. (2000). Control of gammaherpesvirus latency by latent antigen-specific CD8(+) T cells. J Exp Med 192, 943952.
Usherwood, E. J., Ward, K. A., Blackman, M. A., Stewart, J. P. & Woodland, D. L. (2001). Latent antigen vaccination in a model gammaherpesvirus infection. J Virol 75, 82838288.
Virgin, H. W., 4th, Latreille, P., Wamsley, P., Hallsworth, K., Weck, K. E., Dal Canto, A. J. & Speck, S. H. (1997). Complete sequence and genomic analysis of murine gammaherpesvirus 68. J Virol 71, 58945904.[Abstract]
Received 24 October 2003;
accepted 8 December 2003.