Vaccine potential of a murine gammaherpesvirus-68 mutant deficient for ORF73

Polly Fowler and Stacey Efstathiou

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
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
A murine gammaherpesvirus (MHV-68) containing a deletion of the putative plasmid maintenance protein ORF73 exhibits a severe latency deficit. In this study the ability of an ORF73 deletion mutant ({Delta}73) to confer in vivo protection against subsequent challenge with wild-type virus has been examined. Vaccination studies have shown that {Delta}73 vaccination reduced latent infection of wild-type challenge virus to a level below the limit of detection. These results indicate that a live-attenuated gammaherpesvirus that cannot persist is an effective vaccine.


   MAIN TEXT
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
The gammaherpesviruses are characterized by their ability to establish life-long latency in lymphocytes and are commonly associated with lymphoproliferative disorders and malignancies. Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein–Barr virus (EBV) represent important human pathogens which pose a risk in the immunocompromised host. In this setting cellular proliferation is stimulated by the expression of virus-encoded latency-associated gene products and amplification of latently infected cells proceeds unchecked by the immune response. An important goal, therefore, is to develop effective strategies to control, and ultimately protect against latent gammaherpesvirus infection. Due to the species specificity of human gammaherpesviruses it has proved difficult to develop strategies aimed at the prevention and control of latency. For this reason attention has focussed on studying infection of laboratory mice with murine gammaherpesvirus-68 (MHV-68). This tractable system allows potential vaccination strategies to be assessed in terms of their ability to restrict infection and colonization by MHV-68.

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 ({Delta}73) to confer in vivo protection to challenge with wt MHV-68.

The {Delta}73 virus was constructed as described by Fowler et al. (2003). Briefly, 450 bp (genomic co-ordinates, 104 379–104 830; 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. {Delta}73 virus in PBS/1 % fetal calf serum (FCS) or mock-vaccinated with PBS/1 % FCS. To confirm that the vaccinating virus ({Delta}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).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1. Schematic diagram of the vaccination protocol. Five- to six-week-old female BALB/C mice were vaccinated as shown with either 105 p.f.u. in 50 µl PBS/1 % FCS or PBS/1 % FCS alone. Thirty-five or 117 days post-vaccination, mice were challenged with 104 p.f.u. virus in 20 µl PBS/1 % FCS by an i.n. route or with PBS/1 % FCS alone.

 
At 35 days post-vaccination, mice were challenged intranasally (i.n.) with either 104 p.f.u. wt virus or mock-challenged with PBS/1 % FCS. At 4 days post-challenge with wt MHV-68, infectious virus could be detected in the lungs of 5/5 mock-vaccinated animals (Fig. 2a). In contrast, infectious virus was detected in only 2/5 {Delta}73 vaccinated animals and in comparison to mock-vaccinated animals there was a deficit of at least 2 logs in the mean virus titre (Fig. 2a).



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 2. Effect of vaccination on acute infection in the lung. (a) Infectious virus in the lung, mice challenged 35 days post-vaccination. Infectious virus in lung homogenates was assayed on BHK-21 cells after freeze–thaw disruption of tissue 4 days post-challenge. (b) Infectious virus in the lung, mice challenged 117 days post-vaccination. Infectious virus in the lung was assayed as in (a) 5 days post-challenge. Open diamonds, mock vaccine, mock challenge; filled diamonds, mock vaccine, wt challenge; squares, {Delta}73 vaccine, mock challenge; triangles, {Delta}73 vaccine, wt challenge; bars, mean value.

 
To assess the longevity of protection afforded by prior infection with {Delta}73, mice were next challenged 117 days post-vaccination. Infectious virus was assayed 5 days post-challenge and protection against wt virus challenge was still observed; again 1/5 mice had detectable virus in the lung with a 3-log deficit in {Delta}73 vaccinated mice in comparison to mock-vaccinated controls (Fig. 2b). These results suggest that i.p. vaccination with {Delta}73 was able to reduce acute-phase infection in the lung following a high-dose i.n. challenge, and that this protection was maintained up to 117 days post-vaccination.

We then determined the effect of vaccination with {Delta}73 on the establishment and maintenance of latency following wt virus challenge. We first assessed the latent load in splenocytes from mice vaccinated with {Delta}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. 3a). In {Delta}73-vaccinated, wt-challenged mice no infectious centres were detected (Fig. 3a). Similarly, no infectious centres were detected in mice that were {Delta}73-vaccinated and mock-challenged, consistent with previous observations demonstrating that {Delta}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 {Delta}73-vaccinated mice 14 days post-challenge (Fig. 3b).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 3. Effect of vaccination on latency. (a) Latent virus in the spleen in mice vaccinated 35 days prior to challenge. Latent virus was determined by infectious centre assay (Sunil-Chandra et al., 1992) on spleens 14 days post-challenge. Single-cell suspensions of splenocytes were co-cultured with BHK-21 cells for 4–5 days, monolayers were fixed in 10 % formal saline and stained with toluidine blue for enumeration of infectious centres. (b) Latent virus in the spleen in mice vaccinated 117 days prior to challenge. Latent virus was measured by infectious centre assay as for (a) 14 days post-challenge. Symbols are as defined in the legend to Fig. 2.

 
The results from the infectious centre assay indicated effective protection against the establishment of latency after vaccination with the {Delta}73 virus. However, this assay does not distinguish between a reactivation deficit versus a reduction in latent load. Therefore, as a further measure of the degree of protection against latency, we used a limiting-dilution real-time PCR assay (as described by Marques et al., 2003; Fowler et al., 2003) to assess viral load in splenocytes. Using this technique we determined the latent viral load 14 days post-challenge in mice that had been vaccinated 117 days previously. In this assay, real-time PCR primer sets were used within the K3 gene as described previously (Marques et al., 2003; Fowler et al., 2003); for this reason it was not possible to discriminate between vaccine and challenge virus. Therefore, as an internal control for the presence of viral DNA from the vaccine virus, we assessed the genome load in splenocytes from {Delta}73-vaccinated, mock-challenged animals. Consistent with previous findings (Fowler et al., 2003), {Delta}73 genomes could not be detected in this control group of animals, confirming that {Delta}73 has a severe latency deficit. Control reactions to determine the sensitivity of the PCR indicated that the assay was sensitive to 10 copies of K3 in a background of 300 ng cellular DNA (data not shown). Viral genomes were readily detected in the mock-vaccinated, wt-challenged mice; however, viral genomes could not be detected in {Delta}73-vaccinated animals challenged with wt virus (Fig. 4a).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 4. Effect of vaccination on viral load in the spleen. Limiting dilution analysis of viral DNA in vaccinated mice (mice challenged 117 days post-vaccination) was performed as described previously (Marques et al., 2003; Fowler et al., 2003). Fourteen (a) and 57 days (b) post-challenge, single-cell suspensions of splenocytes from five pooled spleens were serially diluted in PBS/2 % FCS after red blood cell lysis. Splenocytes were then lysed overnight and Proteinase K was inactivated by heating to 95 °C for 5 min. Lysates were then analysed for viral DNA by real-time PCR using a LightCycler from Roche Molecular Biochemicals, according to the manufacturer's instructions. Primer-probe sets were used to detect the K3 gene [genome coordinates: upper primer, 24 832–24 850; FL-probe (Roche), 24 973–24 988; LC-probe (Roche), 25 000–25 026; lower primer, 25 049–25 071]. PCR was performed on eight replicates of serial dilutions of splenocytes. Control reactions included titrations of plasmid DNA containing K3 and pHA3 BAC DNA (Adler et al., 2000) in 300 ng naïve cellular DNA and the PCR protocol was shown to be capable of detecting 10 copies of plasmid DNA. A sample of reactions was also checked for integrity of the DNA template by PCR for cellular {beta}-actin, which was readily detectable (data not shown). Symbols are as defined in the legend to Fig. 2.

 
Previously published studies using various vaccination protocols have shown an early, but transient protection from latency (Liu et al., 1999; Stevenson et al., 1999a; Usherwood et al., 2000, 2001). It was therefore important to assess whether the decrease in infectious centres and viral genome load observed at day 14 was maintained at a late time post-challenge. Mice that were challenged with wt virus at 117 days post-vaccination were therefore assessed for latency at 57 days post-challenge, both by infectious centre assay and limiting dilution real-time PCR. At 57 days post-challenge the level of latency was below the limit of detection of the infectious centre assay both in the mock-vaccinated, wt-challenged group, as well as in the {Delta}73-vaccinated animals (data not shown). However, using the more sensitive PCR-based assay, viral genomes were detectable in mock-vaccinated, wt-challenged mice, albeit at a lower level than at 14 days post-challenge (Fig. 4b). In contrast viral genomes were not detected in splenocytes from {Delta}73-vaccinated mice in both the wt- and mock-challenged groups (Fig. 4b). This result indicates that vaccination with the {Delta}73 virus provides long-term protection against the establishment of latency by wt MHV-68.

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 {Delta}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 {beta}-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 {Delta}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 {Delta}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 {Delta}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 {Delta}73 vaccine virus following vaccination, we cannot conclude that the vaccination is sterilizing. It remains possible that there is a low level of persisting {Delta}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 ({Delta}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
 
We would like to thank Philip Stevenson for valuable discussions. This work was supported by an MRC Co-operative Grant (no. G9800943) and The Wellcome Trust (Grant no. 059601). P. F. is supported by a studentship from the Medical Research Council, UK.


   REFERENCES
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Adler, H., Messerle, M., Wagner, M. & Koszinowski, U. H. (2000). Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an infectious bacterial artificial chromosome. J Virol 74, 6964–6974.[Abstract/Free Full Text]

Ballestas, M. E., Chatis, P. A. & Kaye, K. M. (1999). Efficient persistence of extrachromosomal KSHV DNA mediated by latency-associated nuclear antigen. Science 284, 641–644.[Abstract/Free Full Text]

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, 2269–2278.[Abstract/Free Full Text]

Efstathiou, S., Ho, Y. M. & Minson, A. C. (1990). Cloning and molecular characterization of the murine herpesvirus 68 genome. J Gen Virol 71, 1355–1364.[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, 3828–3834.[Abstract/Free Full Text]

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, 3405–3416.[Abstract/Free Full Text]

Kim, I. J., Flano, E., Woodland, D. L. & Blackman, M. A. (2002). Antibody-mediated control of persistent gamma-herpesvirus infection. J Immunol 168, 3958–3964.[Abstract/Free Full Text]

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, 9849–9857.[Abstract/Free Full Text]

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, 7308–7318.[Abstract/Free Full Text]

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, 10295–10303.[Abstract/Free Full Text]

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, 1820–1828.[Abstract/Free Full Text]

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, 5911–5925.[Abstract/Free Full Text]

Stevenson, P. G. & Doherty, P. C. (1998). Kinetic analysis of the specific host response to a murine gammaherpesvirus. J Virol 72, 943–949.[Abstract/Free Full Text]

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, 9281–9286.[Abstract/Free Full Text]

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, 477–483.[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, 3528–3535.[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, 1941–1951.[Abstract/Free Full Text]

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, 3275–3279.[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, 2522–2529.[Abstract/Free Full Text]

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, 943–952.[Abstract/Free Full Text]

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, 8283–8288.[Abstract/Free Full Text]

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, 5894–5904.[Abstract]

Received 24 October 2003; accepted 8 December 2003.