Department of Microbiology, Wake Forest University School of Medicine, Room 5108 Gray Building, Medical Center Boulevard, Winston-Salem, NC 27157, USA1
Author for correspondence: Martha Alexander-Miller. Fax +1 336 716 9928. e-mail marthaam{at}wfubmc.edu
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Recombinant viruses represent powerful vectors for vaccine delivery (reviewed in Cairns & Sarver, 1998 ). A number of positive-strand RNA viruses have been engineered as vaccine vectors to express foreign antigens, including poliovirus (Mandl et al., 1998
; Morrow et al., 1994
), yellow fever virus (McAllister et al., 2000
), Semliki Forest virus (Berglund et al., 1997
) and Venezuelan equine encephalitis virus (Caley et al., 1997
). Very recently, novel approaches towards engineering negative-strand RNA viruses to express foreign genes have also been developed (reviewed in Conzelmann, 1996
). This technology has generated new enthusiasm for exploiting unique features of these RNA viruses for vaccine delivery (Muster et al., 1995
; Roberts et al., 1999
; Schnell et al., 2000
; reviewed in Palese, 1998
). Paramyxoviruses are a group of enveloped negative-sense RNA viruses involved in acute respiratory and systemic infections. This diverse group includes simian virus 5 (SV5), human parainfluenza virus types 14, mumps virus, respiratory syncytial virus (RSV) and measles virus (Lamb & Kolakofsky, 1996
).
In the current study, we have examined the avidity of the CTL elicited using recombinant SV5 (rSV5) as a vaccine vector. rSV5 has excellent potential as a vaccine vector because this non-pathogenic paramyxovirus can infect humans but is not associated with any known disease (Baty et al., 1991 ; Cohn et al., 1996
; Goswami et al., 1984
). In the work reported here, we have engineered an rSV5 to express chicken ovalbumin (Ova), a well-characterized model antigen containing a defined immunodominant epitope in H-2b mice. This model antigen was chosen because the conditions that can identify high and low avidity CTL are well established in our laboratory.
To generate an rSV5 expressing the model antigen Ova, the chicken Ova cDNA (a kind gift from K. Rock, University of Massachusetts Medical Center) (Craiu et al., 1997 ) was inserted between the HN and L genes in the rSV5 cDNA clone (Fig. 1A
). rSV5Ova was generated as described previously (He et al., 1997
) with minor modifications (Parks et al., 2001
).
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To determine how the relative kinetics and level of Ova expression from rSV5 compared with those from rVV, CV-1 cells were infected with rSV5Ova or rVVOva (a kind gift from J. Bennink, NIH) (Restifo et al., 1995 ). At various times post-infection (p.i.), cell lysates were run on a 10% polyacrylamide gel. Proteins were transferred to nitrocellulose and probed using rabbit antibody specific for chicken ovalbumin or the SV5 NP and P proteins (Parks et al., 2001
), followed by HRP-conjugated goat anti-rabbit and ECL. Western blot analysis detected two Ova-specific polypeptides that were not present in mock-infected cells (Fig. 1B
). Endoglycosidase analysis showed that these two forms of Ova were modified by N-linked glycosylation (not shown), but the relationship between the two species has not been established.
In the Western blot shown in Fig. 1(B), the amount of lysate analysed for cells infected with rSV5Ova was twice that of rVVOva. In the case of cells infected with rVVOva, intracellular Ova was detected as early as 3 h p.i. and maximum levels were reached by
15 h p.i. In contrast, significant levels of intracellular Ova were not detected in rSV5Ova-infected cells until
12 h p.i. and the level of Ova was not maximum until 24 h p.i. The expression of the SV5 NP and P proteins was detected earlier than Ova (
69 h p.i.), consistent with the 5' position of the Ova gene and the 3' to 5' gradient of transcription across the SV5 genome.
A pulsechase analysis was carried out to determine the kinetics of Ova synthesis and secretion from cells infected with the two virus vectors. CV-1 cells infected with rSV5Ova or rVVOva were radiolabelled for 15 min at 15 h p.i. using 100 µCi/ml Trans[35S] label, washed in PBS and covered with 0·4 ml of DMEM containing 2% FCS and 2 mM methionine (chase medium). At each time point, medium was removed from the cells, clarified by centrifugation and adjusted to 1% SDS. Likewise, cells were washed with PBS and lysed in 1% SDS. Aliquots from each time point sample were immunoprecipitated using an excess of Ova antiserum as described previously (Erickson & Blobel, 1979 ; Ng et al., 1989
). Cells infected with rSV5Ova synthesized the same or slightly more Ova during the initial radiolabelling period than did cells infected with rVVOva (Fig. 1C
, intracellular lanes). The level of intracellular Ova remained relatively constant in cells infected with rVVOva during the chase period and very little Ova was detected in the extracellular medium. By contrast, the level of intracellular Ova in cells infected with rSV5Ova decreased over time and this was accompanied by a corresponding increase in extracellular Ova (Fig. 1C
, media lanes). Taken together, these data indicated that rVV-infected cells synthesized more Ova at an earlier time after infection than cells infected with rSV5Ova. At later times after infection with rSV5Ova, the level of Ova was very similar to that found in rVVOva-infected cells. Importantly, the non-cytopathic nature of rSV5 infection resulted in a continuous production and secretion of antigen from virus-infected cells compared with rVV infection.
Immunization with vaccinia virus has been shown to elicit a potent CTL response (Kieny et al., 1984 ). In order to compare the CTL response elicited by an rSV5 vector with that elicited by rVV, C57BL/6 mice were anaesthetized by intraperitoneal (i.p.) injection of Avertin followed by intranasal (i.n.) immunization with titrated doses of either rVVOva or rSV5Ova. The number of CTL secreting IFN-
in response to stimulation with the OVA257264 peptide was determined by ELISPOT analysis on day 12 p.i. as previously described (Alexander-Miller, 2000
).
Fig. 2(A) shows that both viral vectors were capable of inducing an Ova-specific CTL response in mice. The dose of rSV5Ova that elicited the largest number of CTL was 1x104 p.f.u. On average, this dose of virus elicited 1·6x104 CTL/spleen. For rVVOva, in some experiments, the highest number of CTL was elicited by immunization with 1 x 102 p.f.u. However, in our hands this dose of virus did not reproducibly induce an immune response, since three of the eight vaccinated mice did not have detectable antiOva CTL (Fig. 2A
; ND, none detected). Therefore 1x103 p.f.u. was chosen as the optimal dose. Using this dose, we also detected an average of approximately 1·6x104 Ova-specific CTL/spleen. Thus, the maximum number of CTL that can be reproducibly elicited by either rSV5Ova or rVVOva is equivalent. Interestingly we found that there was a trend towards decreasing numbers of Ova-specific CTL following immunization with higher doses of rVVOva (1x104 and 1x105 p.f.u.) (Fig. 2A
and data not shown) and to a lesser extent with rSV5Ova (1x105 p.f.u.) (Fig. 2A
). These data therefore suggest that the amount of virus used for i.n. immunization can be a critical parameter in vaccination protocols and the lowest amount of virus that reproducibly gives an immune response should be utilized.
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Mice were immunized with the dose of each virus that yielded equivalent numbers of CTL (1x103 p.f.u. rVVOva and 1x104 p.f.u. rSV5Ova; Fig. 2A) and the Ova-specific CTL response was assayed on day 12. To enumerate high and low avidity cells, splenocytes were cultured in the presence or absence of anti-CD8 antibody (clone 53-6.72). CTL of high avidity are those that produce IFN-
in the presence of anti-CD8 antibody following stimulation with APC pulsed with 10-6 M peptide. CTL of low avidity are those that are blocked by anti-CD8 antibody even in the presence of a high concentration of peptide (10-4 M). Fig. 2(B)
shows the ratio of low to high avidity CTL for mice vaccinated with rVVOva and rSV5Ova, with lower ratios being indicative of more high avidity CTL. The average ratio of low to high avidity CTL generated as a result of immunization with rVVOva was 4·4, while the ratio obtained with rSV5Ova was 2·8. Although the ratios suggested an increase in the elicitation of high avidity CTL in response to rSV5Ova versus rVVOva, the differences were not statistically significant. Thus, these data showed that immunization with rSV5Ova results in CTL with similar avidity to those generated in response to rVVOva.
The previous results showed that during the acute response (day 12 p.i.), the maximum number and avidity of Ova-specific CTL that could be elicited by rSV5Ova and rVVOva was equivalent. However, if a vector is to be considered for use as a vaccine, it is important that it possesses the ability to generate a potent memory response. Thus, we investigated the memory response generated following i.n. immunization with 1 x 103 p.f.u. of rVVOva or 1x104 p.f.u. of rSV5Ova, the doses of virus that yielded equivalent numbers of CTL in the acute response. Using the ELISPOT assay, we initially assessed the total number of Ova-specific CTL present in the memory pool (day 40 p.i.). Fig. 3(A) shows the total number of Ova-specific CTL following immunization with rSV5Ova or rVVOva. The total number of Ova-specific memory CTL generated as a result of immunization with rVVOva was 9·3x103, while 2·8x103 memory CTL were detected following immunization with rSV5Ova (Fig. 3A
). Thus immunization with rVVOva generated approximately 3·3-fold more total Ova-specific CTL. However, when the number of high avidity memory CTL elicited by these vectors was compared, the fold difference was reduced to 2·2 (2·2x103 CTL vs 1·0x103; Fig. 3B
). Thus, low avidity CTL account for a significant percentage of the increased number of memory CTL generated as a result of rVVOva immunization. This is reflected in the data shown in Fig. 3(C)
, where the ratio of low to high avidity memory CTL generated in response to vaccination with rSV5Ova is lower compared with rVVOva (1·6 vs 3·4). Thus, rSV5Ova, while eliciting fewer memory CTL overall, appears to be at least as efficient as rVVOva in generating high avidity CTL for survival into the memory population.
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Previous studies with adoptively transferred cells have demonstrated that high avidity but not low avidity CTL could efficiently reduce virus titres in vivo (Alexander-Miller et al., 1996 ; Gallimore et al., 1998
). The work reported here is the first comparison of the number and functional avidity of the CTL elicited by immunization with a paramyxovirus compared with a vaccinia virus. The rSV5 virus engineered to express a foreign gene (rSV5Ova) reported here elicited a strong high avidity CTL response at day 12 p.i. that was quantitatively and qualitatively similar to that generated in response to a recombinant vaccinia virus (rVVOva) (Figs 2
and 3
). Although the total number of CTL observed in the acute response following immunization with rVVOva or rSV5Ova was equivalent, the total number of CTL surviving into the memory pool following administration of rSV5Ova was somewhat reduced compared with rVVOva. Work is in progress to generate rSV5 vectors that express cytokines that may improve the generation of CTL and their survival into the memory pool.
In previous studies we have analysed the long-term memory response generated following i.p. immunization with rVVOva (Alexander-Miller, 2000 ). In that analysis we found that the ratio of low to high avidity Ova-specific CTL was 2·0 in the acute response compared with 7·1 in the memory compartment. The increased ratio of low to high avidity CTL suggested that following i.p. immunization high avidity CTL survived less efficiently into the memory compartment compared with low avidity CTL. However, in the current study, we did not observe this preferential loss of high avidity CTL in the memory response, as demonstrated by the similar ratio of low to high avidity cells at days 12 and 40. This result was observed following immunization with either rSV5Ova or rVVOva. Although the mechanism responsible for the difference in survival of high avidity Ova-specific CTL following i.p. versus i.n. immunization is unknown, it is possible that an immune response generated in the lung environment differs significantly from that generated at other sites, i.e. in helper cell phenotype (Constant et al., 2000
) or the nature of the APC (Bilyk & Holt, 1993
; Herscowitz, 1985
; Stumbles et al., 1998
). These or other unknown factors may support the preferential generation of high avidity memory CTL following challenge in the lung compared with other sites.
In our titration experiments we found that there was a decrease in the number of detectable CTL in the spleen with high doses of virus (Fig. 2). This was especially evident with rVVOva immunization. The reduction in the number of Ova-specific CTL in our system was also seen in the vaccinia virus-specific CTL response (data not shown). Interestingly, in our previous studies assessing the CTL response induced by vaccinia virus following i.p. immunization, we did not observe a decrease in the number of CTL elicited, even with doses of virus that are 100-fold greater (107 p.f.u.) than the highest dose used here for i.n. immunization (105 p.f.u.) (Alexander-Miller, 2000
, and data not shown). Currently the mechanism responsible for the decrease in CTL is not known. Studies are under way to determine the nature of the loss of antigen-specific activity observed in our system with increasing doses of rVVOva.
In summary, we have found that i.n. immunization with rSV5Ova is capable of eliciting a robust CTL response that is comparable to that observed with rVVOva. Most importantly, vaccination with rSV5Ova was found efficiently to elicit high avidity CTL. This is an extremely desirable attribute of a vaccine vector, as high avidity CTL have been shown to be optimal for virus clearance (Alexander-Miller et al., 1996 ; Derby et al., 2001
). Furthermore, the ability of rSV5 to establish an infection in the respiratory tract should make it useful for elicitation of high avidity CTL specific for respiratory pathogens. The findings reported here support the continued exploration of genetically engineered SV5 as a vaccine vector.
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References |
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Received 6 December 2001;
accepted 10 January 2002.