| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Microbiology and Immunology, Pennsylvania State University, Milton S. Hershey College of Medicine, Hershey, PA 17033, USA
2 Department of Biochemistry and Molecular Biology, The Australian National University, Canberra, ACT, Australia
Correspondence
Christopher C. Norbury
ccn1{at}psu.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Published online ahead of print on 22 June 2007 as DOI 10.1099/vir.0.83107-0.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Nonetheless, significant challenges remain prior to the successful use of recombinant viral vectors to induce protective TCD8+ in humans.
One of the major obstacles to the use of recombinant viruses to produce protective TCD8+ is the phenomenon known as immunodominance. This occurs when TCD8+, responding to an ‘immunodominant’ determinant, suppress the expansion and effector function of TCD8+ specific for other so-called ‘subdominant’ determinants via a variety of mechanisms (Yewdell & Bennink, 1999). When a recombinant viral vector is used, immunodominance is manifested by a strong TCD8+ response targeted to determinants expressed naturally by the vector that reduces or prevents a TCD8+ response to the recombinantly encoded foreign antigen. For example, recombinant vaccinia virus (rVACV) shows a reduced efficacy at inducing TCD8+ to transgene-encoded antigens compared with the pathogens that encode the antigen naturally (Harrington et al., 2002). In a recent study using a VACV-based cancer vaccine in humans, the dominant TCD8+ response was directed against VACV proteins, rather than against the recombinantly encoded tumour antigen (Smith et al., 2005). Although it is possible in experimental systems to remove immunodominant determinants in order to enhance the response to subdominant antigens (Tanaka et al., 1989; Webby et al., 2003; Weidt et al., 1998), this approach is not practical in a therapeutic setting, as there are no means to predict the immunodominance of antigens in an outbred population.
In this study, we have successfully employed a strategy that reduces immunodominance following immunization with VACV vectors. This was accomplished by inhibiting vector gene expression by using psoralen and UV irradiation. UV/psoralen has been suggested as an inactivating agent for vaccines (Brockstedt et al., 2005) and its use is well established as an experimental tool for studying vaccinia virus (Tsung et al., 1996). DNA damage induced by UV/psoralen is random and smaller genes are therefore inactivated less frequently. Here, we exploit this property of psoralen/UV by using recombinant VACV vaccines that express minimal antigenic-peptide ‘minigenes’ (8–10 aa). Minigenes are small targets for irradiation-induced DNA damage, allowing broad ablation of VACV gene expression while minigene expression remains relatively intact. We show that the resulting VACV vector is non-replicating and elicits less inflammation, but induces more foreign epitope-specific TCD8+ than untreated virus.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Viruses and cells.
rVACVs were made by homologous recombination (Chakrabarti et al., 1985) and have been described previously (Anton et al., 1999; Gould et al., 1989; Restifo et al., 1995; Yewdell et al., 1985, 1986). The control rVACV used expressed 
-galactosidase (-gal) driven by the p11 promoter, as do the other rVACVs used in this study (Chakrabarti et al., 1985). WT3 cells (Pretell et al., 1979) were grown in Dulbecco's modified Eagle's medium (Invitrogen) with 5 % fetal bovine serum (FBS). B3Z 1.G7D7 T-cell hybridoma cells (Sanderson & Shastri, 1994) were subcloned and grown in RPMI 1640 medium (Invitrogen) with 5 % FBS.
Peptides.
Antigenic peptides for vesicular stomatitis virus N52–59 (RGYVYQGL), influenza A virus (IAV) PR8 NP366–374 (ASNENMETM), VACV B8R20–27 (TSYKFESV), VACV A47L138–146 (AAFEFINSL), VACV K3L6–15 (YSLPNAGDVI), VACV A42R88–96 (YAPVSPIVI), VACV A19L47–55 (VSLDYINTM) and chicken egg ovalbumin (OVA)257–264 (SIINFEKL) were synthesized by the Macromolecule Core Facility at the Hershey Medical Center or purchased from Mimotopes (Fisher Research).
Intracellular cytokine staining (ICS).
Female C57BL/6 mice, 6–8 weeks of age, were immunized intraperitoneally (i.p.) or intravenously (i.v.) with 0.5x106 p.f.u. virus (using pre-treatment values). After 6 days, spleens were harvested and homogenized. Lymphocytes were isolated by centrifugation over lymphocyte separation medium (Cambrex) and then stimulated for 6 h at 37 °C with WT3 cells that were either virus-infected for 4 h or pulsed at 25 °C for 40 min with 1 µM peptide (Ljunggren et al., 1990). Two hours into the stimulation, brefeldin A (Sigma) was added to a concentration of 5 µg ml–1. Lymphocytes were then incubated on ice for 15 min in supernatant from the 2.4G2 hybridoma containing 10 % normal mouse serum (Sigma). Cells were stained with phycoerythrin–Cy5-conjugated anti-CD8
antibody (clone 53-6.7; BD Pharmingen) for 40 min on ice, then fixed with 1 % paraformaldehyde for 10 min at 25 °C. Lymphocytes were stained with fluorescein isothiocyanate-conjugated anti-mouse gamma interferon (IFN-
) antibody (clone XMG1.2; BD Pharmingen) in Fc block/0.5 % saponin (Sigma) for 40 min on ice. Flow cytometry was used to determine the percentage of CD8+ lymphocytes producing IFN-. Background levels of staining in the absence of stimulation were subtracted to obtain final values. In all experiments involving ICS, immunization with rVACV treated with psoralen and no UV induced levels of specific TCD8+ comparable to those with untreated rVACV. Data shown are representative of several repeats of each experiment.
UV/psoralen treatment of viruses.
4,5',8-Trimethylpsoralen (Sigma) was added to 1 ml rVACV stock (1x108 p.f.u. ml–1) to give a final concentration of 10 µg ml–1. The stock was then exposed to UV-C (254 nm) for various times by placing a portable UV lamp approximately 1 cm above the liquid.
Quantification of cell-surface peptide–major histocompatibility complex (MHC) class I complexes.
WT3 cells were infected with treated or untreated rVACV for 4 h. Cells were washed twice in Iscove's modified Dulbecco's medium (Invitrogen) with 10 % FBS and incubated for 15 min in Fc block. Cells were then stained with Alexa Fluor 647-conjugated antibody specific for the SIINFEKL–Kb complex (25D1.16) (Porgador et al., 1997). Fluorescence was analysed by flow cytometry.
Activation of OVA-specific T-cell hybridoma.
WT3 cells were infected with treated or untreated rVACV in the presence of 40 µg cytosine arabinoside (Sigma) ml–1 and co-incubated at 37 °C/5 % CO2 with B3Z T-cell hybridomas at a ratio of 1 : 1. After 24 h, a -gal substrate that contained 0.125 % Igepal and chlorophenol red--D-galactopyranoside (Sigma) was added to the cells (Malarkannan et al., 2001). Plates were incubated at 37 °C until a colour change was apparent (30–45 min); the reaction was then halted by adding 300 mM glycine and 15 mM EDTA at pH 12.0 (Malarkannan et al., 2001). A570 was determined with an MRX plate reader.
Measurement of inflammation.
Mice were immunized intradermally (i.d.) in each ear pinna with 10 µl 1.0x106 p.f.u. VACV ml–1 suspended in Hanks' balanced salt solution (HBSS)/0.1 % BSA. We used wild-type VACV Western Reserve rather than the recombinant viruses used elsewhere in the study. The insertion of genes by homologous recombination causes deletion of the thymidine kinase gene, which reduces the in vivo replication of viruses (Buller et al., 1985) and dramatically reduces the induction of inflammation induced in vivo. Ear thickness was measured at 24 h intervals by using a micrometer (Mitutoyo America Corp.). Swelling in the ears of mice immunized with untreated VACV was comparable to that of mice immunized with VACV that were treated with psoralen and no UV.
Analysis of late gene expression.
WT3 cells were infected with either VSC8 (-gal driven by the p11 promoter) or VLW (-gal driven by the p7.5 promoter) that had been treated with psoralen and exposed to UV-C for varying lengths of time. After 7 h, -gal expression was quantified with chlorophenol red--D-galactopyranoside as outlined above.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). C57BL/6 mice were immunized i.p. with VSV, rVACV encoding the full-length VSV N, rVACV encoding the VSV N52–59 minimal antigenic determinant or a control rVACV. After 6 days, splenocytes were isolated and stimulated with VSV-infected cells (which are able to present the entire repertoire of H-2b-restricted VSV determinants), VSV N52–59-pulsed cells (which present only the VSV N52–59 determinant) or control rVACV-infected cells (which are able to present the entire repertoire of H-2b VACV determinants). IFN- production in response to peptide-pulsed or virus-infected cells was determined by using an ICS assay.
Immunization with VSV itself was significantly more efficient (8–9-fold higher percentage of TCD8+) at producing both VSV-specific and VSV N52–59-specific TCD8+ than immunization with rVACV (Fig. 1a, b). In contrast, 2–3-fold greater numbers of VACV-specific TCD8+ were induced compared with VSV-specific or N52–59-specific TCD8+ following immunization with rVACV encoding the full-length VSV N (Fig. 1c). Thus, rVACV immunization remains markedly less efficient at inducing a specific TCD8+ response than immunization with a virus expressing antigen in its natural context.
|
Similar to the results seen with VSV, i.p. immunization with IAV was more efficient at producing NP366–374-specific TCD8+ than immunization with rVACV expressing either full-length NP or the NP366–374 minigene (Fig. 1d, e
). Similar to previous reports (Harrington et al., 2002), the numbers of TCD8+ specific for VACV determinants were up to 30-fold higher than the numbers of TCD8+ specific for the recombinant antigen (Fig. 1f). Similar results were observed for TCD8+ cells isolated by peritoneal lavage 6 days post-immunization and for splenic memory TCD8+ responses measured 4 weeks post-immunization (data not shown). These results indicate that the induction of a TCD8+ response to the same antigen is markedly less efficient when it is expressed during rVACV infection than when expressed in its natural context.
Ablation of viral transcription by using UV/psoralen treatment
We reasoned that rVACV vectors in which viral gene expression is inhibited while foreign antigen expression is maintained will have an advantage as a vaccine, as they will induce fewer immunodominant TCD8+. To achieve such a vaccine, we used an rVACV expressing a foreign antigen as a minigene and inactivated viral gene expression by using UV/psoralen treatment. This method will be effective only if there is a dose of treatment that will broadly reduce VACV gene expression while leaving expression of the minigene (which represents a much smaller target for DNA damage) intact.
Long-wave UV/psoralen treatment has previously been shown to ablate VACV genes in a size-dependent manner (Tsung et al., 1996). To test whether UV-C/psoralen-mediated inhibition of gene expression is similarly dependent on the size of a gene, we used two rVACVs that express the green fluorescent protein (GFP). The first rVACV expresses the chimeric protein NP–GFP (Anton et al., 1999) (744 aa, >2.2 kb) and the second expresses the shorter GFP alone (237 aa, 0.7 kb). Virus stocks were subjected to UV/psoralen at a range of doses and GFP fluorescence was measured by flow cytometry after 4 h infection of WT3 cells. Expression of the larger NP–GFP was almost undetectable in infected cells when the virus was treated with 10 µg psoralen ml–1 and irradiated for 20 s with UV-C (Fig. 2a). In contrast, GFP expression was readily detectable, although significantly reduced, after 60 s exposure to UV-C (Fig. 2a). These results demonstrate that, by choosing dose carefully, UV/psoralen can be used to inhibit the expression of large VACV-encoded genes selectively, while leaving the expression of shorter genes relatively intact.
|
; Princiotta et al., 2003). As expected, much higher levels of surface SIINFEKL–Kb complexes were generated after infection with rVACV-OVA257–264 than with rVACV-OVA, due to the larger quantities of relevant peptide produced after infection with the former, in addition to the requirement for proteasomal processing of the latter antigen. As little as 40 s UV-C treatment of rVACV-OVA reduced the levels of SIINFEKL–Kb complexes to background levels (Fig. 2b). In contrast, after infection with rVACV-OVA257–264 treated with 40 s UV-C, surface SIINFEKL–Kb levels were still greater than those generated from untreated rVACV-OVA. Indeed, SIINFEKL–Kb complexes could be detected on the surface of cells infected with rVACV-OVA257–264 treated with psoralen and UV-C for 120 s (data not shown).
Although 25D1.16 staining is a direct measurement of peptide–MHC presentation, T-cell activation may occur at levels of surface peptide–MHC that are undetectable using this antibody (Porgador et al., 1997). To assess antigen presentation to T cells, we examined the ability of virus-infected cells to trigger the SIINFEKL-specific lacZ T-cell hybridoma B3Z. The B3Z hybridoma expresses the lacZ gene driven by the interleukin-2 response element NFAT promoter, so -gal is produced when the cells are stimulated through the T-cell receptor (Sanderson & Shastri, 1994). WT3 cells were infected with rVACV-OVA or rVACV-OVA257–264 that had been treated with psoralen and varying doses of UV-C. Infected WT3 cells were incubated overnight with the B3Z T-cell hybridoma, and antigen presentation was measured the following day by using a colorimetric substrate of -gal.
B3Z T-cell hybridomas detected SIINFEKL–Kb complexes with kinetics similar to those for 25D1.16 staining, but presentation could be detected after treatment of virus with UV-C and psoralen for up to 5 min (data not shown). T-cell activation by WT3 cells infected with rVACV-OVA is detectable at low levels after pre-treatment of virus with psoralen and 40 s exposure to UV-C, but is ablated completely after 60 s UV-C exposure (Fig. 2c). Thus, MHC class I-restricted presentation of minigene is preserved under conditions that ablate presentation of peptides derived from longer proteins.
UV/psoralen treatment enhances in vivo TCD8+ responses to rVACV-encoded minigenes, but inhibits responses to native VACV epitopes
To test whether UV/psoralen-treated rVACV could induce primary TCD8+ to an encoded minigene, mice were immunized i.v. with rVACV-OVA257–264 treated with psoralen and increasing doses of UV-C. Six days later, splenic responses to VACV-infected cells and SIINFEKL peptide were measured by using ICS for IFN-. The percentage of VACV-specific TCD8+ decreased with increasing doses of UV-C, with a >66 % decrease when rVACV-OVA257–264 was exposed to UV-C for 1 min (Fig. 3a). In contrast, the percentage of SIINFEKL-specific TCD8+ increased with UV/psoralen treatment of rVACV-OVA257–264, reaching a >8-fold increase with 1 min treatment (Fig. 3b). Immunization with UV/psoralen-treated VACV results in a lesser expansion in the cellularity of the spleen than immunization with VACV treated with psoralen alone (Fig. 3c). However, when the reduced splenic cellularity was taken into account, the increase in the percentage of SIINFEKL-specific TCD8+ represented a 3-fold increase in the total number of these cells (Fig. 3d). To investigate whether the increase in SIINFEKL-specific TCD8+ was related to the size of a target antigen gene for psoralen incoporation, we also immunized mice with UV/psoralen-treated VACV expressing full-length OVA. When SIINFEKL was encoded in the context of the full-length OVA gene, as little as 1 min UV-C/psoralen treatment was able to ablate the specific TCD8+ response to this determinant (Fig. 3e), indicating that the enhanced response was due to the small size of the SIINFEKL minigene.
|
), allowing us to test the effect of UV/psoralen treatment on priming of TCD8+ to individual vector epitopes. These epitopes are located at various distances from the amino terminus of their protein, ranging from as few as 6 to 138 aa. Four of these determinants, namely B8R20–27, K3L6–15, A47L138–146 and A19L47–55, are derived from the products of genes classified as early. Only one, A42R88–96, is derived from the product of a gene classified as late. In the experiment described above, lymphocytes were also stimulated with peptide-pulsed cells and the response to each of the VACV epitopes was quantified similarly by using ICS for IFN-.
The response to four of the five VACV determinants decreased or remained stable at low levels upon UV/psoralen treatment, with the exception of K3L6–15 (Fig. 4a). Similar to a minigene, the response to this peptide increased with UV/psoralen treatment, with a maximum response occurring after 1 min exposure to UV-C. This result is not unexpected, because the epitope is only 6 aa from the amino terminus of the K3L protein and so will probably act in a manner resembling the similarly sized minigene. The sum of the number of TCD8+ responding to the five known VACV determinants was compared with the number of TCD8+ responding to VACV-infected cells. The TCD8+ response to the VACV determinants did not decrease as rapidly as that to VACV-infected cells (Fig. 4b), indicating that the entire VACV response may act differently from that of the mapped determinants.
|
) and bacteria (Brockstedt et al., 2005), and has been used to attenuate rVACV vaccine vectors in other studies (Oertli et al., 1996; Zajac et al., 1997) by blocking late gene transcription. To verify that our UV-C/psoralen treatment also inhibits late gene expression, we used rVACVs that express -gal driven either by the strict late p11 promoter or the early/late p7.5 promoter. Virus stocks were treated with psoralen and increasing doses of UV-C, and functional -gal production was measured after 7 h infection of WT3 cells. UV-C treatment for 10 s reduced -gal expression from the strict late p11 promoter to background levels, whilst reducing expression from the early/late promoter by only 25 % (Fig. 5a). Ablation of -gal production driven by p7.5 required 30 s UV treatment. We next assessed the ability of UV/psoralen-treated virus to generate plaques in a standard titration assay. The replicative competence of the rVACV in our studies was compromised severely after 30 s UV treatment (Fig. 5b) and there were no observable plaques following as little as 1 min UV/psoralen treatment (data not shown).
|
). Reduction or ablation of replication has been linked to a significant reduction in the inflammatory response induced in vivo (Sutter et al., 1994). To assess whether this was the case following UV/psoralen treatment, we infected mice i.d. in each ear pinna (Tscharke et al., 2002) with VACV that was treated with psoralen and 0 or 1 min UV-C. Ear thickness was then measured daily to gauge the severity of the resulting inflammatory response.
Following challenge with 104 p.f.u. VACV, ear thickness increased rapidly in mice infected with VACV that had been treated with psoralen and no UV-C. Ear thickness continued to increase until 5 days post-injection, when a mean increase of 0.7 mm was measured (Fig. 6). After 6 days, the swelling did not subside, but the injected ears displayed extensive scabbing at the inoculation site and substantial necrosis was apparent. In contrast, mice inoculated with VACV treated with psoralen and 1 min UV-C treatment displayed no measurable increase in ear thickness (Fig. 6) and were comparable to mice injected with HBSS only (data not shown).
|
). Thus, UV/psoralen-treated VACV induces a markedly reduced inflammatory response in vivo following either i.d. or i.v. immunization compared with untreated VACV. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Previously, treatment with psoralen and long-wave UV-A has been used to ablate the replication (Hanson et al., 1978) and cytopathic effects (Tsung et al., 1996) of VACV. This treatment blocks transcription of late genes, but not early genes, and produces a non-replicating virus that is significantly attenuated and safer to use as a vaccine vector (Oertli et al., 1996; Zajac et al., 1997), as it can neither spread from the site of infection nor initiate a severe inflammatory response. Inflammatory complications and unrestrained growth of the virus following immunization are one of the leading obstacles to the use of VACV as a vector, particularly in immunocompromised populations. Here, we exploit the cross-linking efficacy of short-wave UV-C to ablate production of a large number of intact VACV proteins encoded by both early and late genes. By encoding recombinant antigen as a minigene, the resulting drop in the efficiency of antigen presentation is functionally irrelevant, as the minigene produces much higher levels of peptide–MHC complexes in infected cells than those generated from rVACV encoding full-length antigen.
The increase in TCD8+ response to a minigene after broad inhibition of VACV gene expression is probably due to two mechanisms acting in a cooperative manner. First, ablation of VACV gene expression reduced the induction of VACV-specific TCD8+, freeing minigene-specific TCD8+ from immunodomination by VACV-specific TCD8+. In other systems, removal of an immunodominant determinant leads to an increase in the frequency of TCD8+ specific for subdominant determinants (Tanaka et al., 1989; Webby et al., 2003; Weidt et al., 1998). By treating rVACV with UV/psoralen, we have decreased the production of viral proteins, the source of all immunodominant determinants, regardless of the genetic background of the organism being immunized. The resulting increase in the percentage of TCD8+ specific for the OVA257–264 determinant can be reproduced after immunization with rVACV encoding minigenes derived from both VSV N protein and Sendai virus nucleoprotein (data not shown). Reduced immunodominance probably acts in concert with the ablation of the production of VACV immunomodulators by UV/psoralen treatment. Genetic removal of two such genes from VACV has been shown to increase the magnitude of the VACV-specific TCD8+ response (Clark et al., 2006; Staib et al., 2005) and would probably increase the response to an encoded minigene.
As little as 1 min UV/psoralen treatment reduced the entire VACV-specific TCD8+ response by as much as 66 %, a much greater decrease than that seen in the sum of the five VACV determinants mapped to date (Fig. 4b). In fact, these five determinants comprise only about 40 % of the entire response in H-2b mice (Tscharke et al., 2005). Thus, the decrease in the TCD8+ response to VACV-infected cells probably results from UV/psoralen treatment-mediated ablation of hitherto-undiscovered VACV encoded determinant(s).
UV/psoralen treatment for 1 min that produces a 66 % reduction of the VACV-specific TCD8+ response probably results from the targeting of two groups of determinants. The primary targets of UV/psoralen treatment are those determinants encoded within late genes. One of the determinants to which the TCD8+ response was relatively stable after 5 min UV/psoralen treatment, A42R88–96, is a product of a gene that has been classified as late (Blasco et al., 1991). Late gene transcription was blocked under conditions (10 µg psoralen ml–1, 1 min UV-C) that were still able to generate a TCD8+ response to the A42R determinant (Fig. 5a). Thus, either this determinant is generated from protein in the viral stocks, an unlikely scenario as increasing UV treatment to longer than 20 min ablates the response (data not shown), or the A42R gene is not a strict late gene. In the initial characterization of this VACV gene, low levels of protein were generated in the presence of cytosine arabinoside, an analogue of deoxycytidine that blocks DNA replication and thus late gene production (Blasco et al., 1991). Mapping of VACV-encoded determinants in late genes has typically been disregarded, as recombinant antigens driven by late promoters often fail to induce antigen-specific TCD8+ following immunization (Bronte et al., 1997; Coupar et al., 1986). However, our data indicate that viral genes may not always fall into easily distinguishable categories, and disregarding groups of genes as non-immunogenic may produce misleading results.
In addition to a direct effect upon responses to late VACV genes, UV/psoralen treatment will also probably target antigenic determinants present at sites further from the initiation of genes in which they are encoded. The TCD8+ response to a determinant encoded by the K3L gene (K3L6–15) increased significantly upon UV/psoralen treatment, with the number of responding cells peaking at 1 min UV-C exposure and declining thereafter (Fig. 4a). The kinetics of this increase were similar to those observed with the response to the encoded OVA257–264 minigene (Fig. 3b). K3L6–15 is very close to the amino terminus of the protein, making psoralen incorporation between the start codon and the coding sequence of the determinant very unlikely. Thus, expression of this determinant following UV/psoralen treatment is probably preserved in a similar way to the recombinant minigene determinant. In contrast, the A47L138–146 determinant is located furthest from the start codon and the TCD8+ response to this determinant was ablated completely upon 1 min UV/psoralen treatment.
Decreased inflammation following immunization with UV/psoralen-treated rVACV is an attractive characteristic from a vaccine perspective. Attenuated VACV strains, such as modified virus Ankara (MVA), are commonly used as vaccine vectors and have been shown to induce a milder inflammatory response than wild-type VACV (Stickl & Hochstein-Mintzel, 1971). However, these attenuated strains are still limited by many of the other drawbacks of wild-type VACV when used as a vaccine vector. MVA elicits a robust humoral response (Jones-Trower et al., 2005) that can prevent reimmunization and, despite deletions in large regions of the genome, MVA still encodes many immunomodulatory genes that can blunt a TCD8+ response to a recombinant antigen. Following immunization with UV/psoralen-treated rVACV, we observed a dramatic (16-fold) reduction in the titres of neutralizing antibody (data not shown). In addition, UV/psoralen treatment of VACV can ablate production of virus-encoded immunomodulatory molecules. Finally, MVA still elicits large TCD8+ responses that can be immunodominant and suppress the desired TCD8+ response to a recombinant antigen (Smith et al., 2005). It is possible that the reduction of vector gene expression in attenuated VACV strains such as MVA could improve the efficacy of these vectors as vaccines.
The strategy outlined here of selectively targeting VACV gene expression reduces vector immunodominance and expression of immunomodulators, leading to an increased frequency of TCD8+ specific for the recombinantly encoded minigene. The minigene-specific memory TCD8+ response under these circumstances is similar in magnitude to that observed after immunization with replicating VACV (data not shown). Thus, production of a potential vector of equal or greater efficacy that induces significantly reduced levels of anti-VACV neutralizing antibody and dramatically reduced inflammatory responses points a way to a safe and effective vaccination strategy. Overall, we have demonstrated that the reduction of VACV vector expression allows the more effective generation of antigen-specific TCD8+ following immunization.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Blasco, R., Cole, N. B. & Moss, B. (1991). Sequence analysis, expression, and deletion of a vaccinia virus gene encoding a homolog of profilin, a eukaryotic actin-binding protein. J Virol 65, 4598–4608.
Brockstedt, D. G., Bahjat, K. S., Giedlin, M. A., Liu, W., Leong, M., Luckett, W., Gao, Y., Schnupf, P., Kapadia, D. & other authors (2005). Killed but metabolically active microbes: a new vaccine paradigm for eliciting effector T-cell responses and protective immunity. Nat Med 11, 853–860.[CrossRef][Medline]
Bronte, V., Carroll, M. W., Goletz, T. J., Wang, M., Overwijk, W. W., Marincola, F., Rosenberg, S. A., Moss, B. & Restifo, N. P. (1997). Antigen expression by dendritic cells correlates with the therapeutic effectiveness of a model recombinant poxvirus tumor vaccine. Proc Natl Acad Sci U S A 94, 3183–3188.
Buller, R. M., Smith, G. L., Cremer, K., Notkins, A. L. & Moss, B. (1985). Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype. Nature 317, 813–815.[CrossRef][Medline]
Chakrabarti, S., Brechling, K. & Moss, B. (1985). Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques. Mol Cell Biol 5, 3403–3409.
Clark, R. H., Kenyon, J. C., Bartlett, N. W., Tscharke, D. C. & Smith, G. L. (2006). Deletion of gene A41L enhances vaccinia virus immunogenicity and vaccine efficacy. J Gen Virol 87, 29–38.
Coupar, B. E., Andrew, M. E., Both, G. W. & Boyle, D. B. (1986). Temporal regulation of influenza hemagglutinin expression in vaccinia virus recombinants and effects on the immune response. Eur J Immunol 16, 1479–1487.[Medline]
Fremont, D. H., Matsumara, M., Stura, E. A., Peterson, P. A. & Wilson, I. A. (1992). Crystal structures of two viral peptides in complex with murine MHC class I H-2Kb. Science 257, 919–926.
Gould, K., Cossins, J., Bastin, J., Brownlee, G. G. & Townsend, A. (1989). A 15 amino acid fragment of influenza nucleoprotein synthesized in the cytoplasm is presented to class I-restricted cytotoxic T lymphocytes. J Exp Med 170, 1051–1056.
Hanson, C. V., Riggs, J. L. & Lennette, E. H. (1978). Photochemical inactivation of DNA and RNA viruses by psoralen derivatives. J Gen Virol 40, 345–358.
Harrington, L. E., Most Rv, R., Whitton, J. L. & Ahmed, R. (2002). Recombinant vaccinia virus-induced T-cell immunity: quantitation of the response to the virus vector and the foreign epitope. J Virol 76, 3329–3337.
Jones-Trower, A., Garcia, A., Meseda, C. A., He, Y., Weiss, C., Kumar, A., Weir, J. P. & Merchlinsky, M. (2005). Identification and preliminary characterization of vaccinia virus (Dryvax) antigens recognized by vaccinia immune globulin. Virology 343, 128–140.[CrossRef][Medline]
Lane, J. M., Ruben, F. L., Neff, J. M. & Millar, J. D. (1969). Complications of smallpox vaccination, 1968. N Engl J Med 281, 1201–1208.[Medline]
Ljunggren, H. G., Stam, N. J., Ohlen, C., Neefjes, J. J., Hoglund, P., Heemels, M. T., Bastin, J., Schumacher, T. N., Townsend, A. & other authors (1990). Empty MHC class I molecules come out in the cold. Nature 346, 476–480.[CrossRef][Medline]
Malarkannan, S., Mendoza, L. M. & Shastri, N. (2001). Generation of antigen-specific, lacZ-inducible T-cell hybrids. Methods Mol Biol 156, 265–272.[Medline]
Oertli, D., Marti, W. R., Norton, J. A. & Tsung, K. (1996). Non-replicating recombinant vaccinia virus encoding murine B-7 molecules elicits effective costimulation of naive CD4+ splenocytes in vitro. J Gen Virol 77, 3121–3125.
Porgador, A., Yewdell, J. W., Deng, Y., Bennink, J. R. & Germain, R. N. (1997). Localization, quantitation, and in situ detection of specific peptide-MHC class I complexes using a monoclonal antibody. Immunity 6, 715–726.[CrossRef][Medline]
Pretell, J., Greenfield, R. S. & Tevethia, S. S. (1979). Biology of simian virus 40 (SV40) transplantation antigen (TrAg). V. In vitro demonstration of SV40 TrAg in SV40 infected nonpermissive mouse cells by the lymphocyte mediated cytotoxicity assay. Virology 97, 32–41.[CrossRef][Medline]
Princiotta, M. F., Finzi, D., Qian, S. B., Gibbs, J., Schuchmann, S., Buttgereit, F., Bennink, J. R. & Yewdell, J. W. (2003). Quantitating protein synthesis, degradation, and endogenous antigen processing. Immunity 18, 343–354.[CrossRef][Medline]
Restifo, N. P., Bacik, I., Irvine, K. R., Yewdell, J. W., McCabe, B. J., Anderson, R. W., Eisenlohr, L. C., Rosenberg, S. A. & Bennink, J. R. (1995). Antigen processing in vivo and the elicitation of primary CTL responses. J Immunol 154, 4414–4422.[Abstract]
Sanderson, S. & Shastri, N. (1994). LacZ inducible, antigen/MHC-specific T cell hybrids. Int Immunol 6, 369–376.
Smith, C. L., Mirza, F., Pasquetto, V., Tscharke, D. C., Palmowski, M. J., Dunbar, P. R., Sette, A., Harris, A. L. & Cerundolo, V. (2005). Immunodominance of poxviral-specific CTL in a human trial of recombinant-modified vaccinia Ankara. J Immunol 175, 8431–8437.
Staib, C., Kisling, S., Erfle, V. & Sutter, G. (2005). Inactivation of the viral interleukin 1 receptor improves CD8+ T-cell memory responses elicited upon immunization with modified vaccinia virus Ankara. J Gen Virol 86, 1997–2006.
Stickl, H. & Hochstein-Mintzel, V. (1971). Intracutaneous smallpox vaccination with a weak pathogenic vaccinia virus ("MVA virus"). Munch Med Wochenschr 113, 1149–1153 (in German)[Medline]
Sutter, G., Wyatt, L. S., Foley, P. L., Bennink, J. R. & Moss, B. (1994). A recombinant vector derived from the host range-restricted and highly attenuated MVA strain of vaccinia virus stimulates protective immunity in mice to influenza virus. Vaccine 12, 1032–1040.[CrossRef][Medline]
Tanaka, Y., Anderson, R. W., Maloy, W. L. & Tevethia, S. S. (1989). Localization of an immunorecessive epitope on SV40 antigen by H-2Db-restricted cytotoxic T-lymphocyte clones and a synthetic peptide. Virology 171, 205–213.[CrossRef][Medline]
Truckenmiller, M. E. & Norbury, C. C. (2004). Viral vectors for inducing CD8+ T cell responses. Expert Opin Biol Ther 4, 861–868.[CrossRef][Medline]
Tscharke, D. C., Reading, P. C. & Smith, G. L. (2002). Dermal infection with vaccinia virus reveals roles for virus proteins not seen using other inoculation routes. J Gen Virol 83, 1977–1986.
Tscharke, D. C., Karupiah, G., Zhou, J., Palmore, T., Irvine, K. R., Haeryfar, S. M., Williams, S., Sidney, J., Sette, A. & other authors (2005). Identification of poxvirus CD8+ T cell determinants to enable rational design and characterization of smallpox vaccines. J Exp Med 201, 95–104.
Tsung, K., Yim, J. H., Marti, W., Buller, R. M. L. & Norton, J. A. (1996). Gene expression and cytopathic effect of vaccina virus inactivated by psoralen and long-wave UV light. J Virol 70, 165–171.[Abstract]
Webby, R. J., Andreansky, S., Stambas, J., Rehg, J. E., Webster, R. G., Doherty, P. C. & Turner, S. J. (2003). Protection and compensation in the influenza virus-specific CD8+ T cell response. Proc Natl Acad Sci U S A 100, 7235–7240.
Weidt, G., Utermohlen, O., Heukeshoven, J., Lehmann-Grubbe, F. & Deppert, W. (1998). Relationship among immunodominance of single CD8+ T cell epitopes, virus load, and kinetics of primary antiviral CTL response. J Immunol 160, 2923–2931.
Yewdell, J. W. & Bennink, J. R. (1999). Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses. Annu Rev Immunol 17, 51–88.[CrossRef][Medline]
Yewdell, J. W., Bennink, J. R., Smith, G. L. & Moss, B. (1985). Influenza A virus nucleoprotein is a major target antigen for cross- reactive anti-influenza A virus cytotoxic T lymphocytes. Proc Natl Acad Sci U S A 82, 1785–1789.
Yewdell, J. W., Bennink, J. R., Mackett, M., Lefrancois, L., Lyles, D. S. & Moss, B. (1986). Recognition of cloned vesicular stomatitis virus internal and external gene products by cytotoxic T lymphocytes. J Exp Med 163, 1529–1538.
Zajac, P., Oertli, D., Spagnoli, G. C., Noppen, C., Schaefer, C., Heberer, M. & Marti, W. R. (1997). Generation of tumoricidal cytotoxic T lymphocytes from healthy donors after in vitro stimulation with a replication-incompetent vaccinia virus encoding MART-1/Melan-A 27–35 epitope. Int J Cancer 71, 491–496.[CrossRef][Medline]
Received 20 April 2007;
accepted 13 June 2007.
This article has been cited by other articles:
|
|
 |
|
  M. J. Palmowski, M. Parker, K. Choudhuri, C. Chiu, M. F. C. Callan, P. A. van der Merwe, V. Cerundolo, and K. G. Gould A Single-Chain H-2Db Molecule Presenting an Influenza Virus Nucleoprotein Epitope Shows Enhanced Ability at Stimulating CD8+ T Cell Responses In Vivo J. Immunol., April 15, 2009; 182(8): 4565 - 4571. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Jing, D. H. Davies, T. M. Chong, S. Chun, C. L. McClurkan, J. Huang, B. T. Story, D. M. Molina, S. Hirst, P. L. Felgner, et al. An Extremely Diverse CD4 Response to Vaccinia Virus in Humans Is Revealed by Proteome-Wide T-Cell Profiling J. Virol., July 15, 2008; 82(14): 7120 - 7134. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bansal, B. Jackson, K. West, S. Wang, S. Lu, J. S. Kennedy, and P. A. Goepfert Multifunctional T-Cell Characteristics Induced by a Polyvalent DNA Prime/Protein Boost Human Immunodeficiency Virus Type 1 Vaccine Regimen Given to Healthy Adults Are Dependent on the Route and Dose of Administration J. Virol., July 1, 2008; 82(13): 6458 - 6469. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
) or GFP (
) were treated with psoralen and irradiated with UV-C for the periods of time shown. Fluorescent gene production was measured by flow cytometry following infection of WT3 cells. (b, c) WT3 cells were infected in vitro with either rVACV-OVA257–264 (
