High avidity cytotoxic T lymphocytes to a foreign ... - Semantic Scholar

Journal of General Virology (2002), 83, 1167–1172. Printed in Great Britain ..........................................................................................................................................................................................................

SHORT COMMUNICATION

High avidity cytotoxic T lymphocytes to a foreign antigen are efficiently activated following immunization with a recombinant paramyxovirus, simian virus 5 Griffith D. Parks and Martha A. Alexander-Miller Department of Microbiology, Wake Forest University School of Medicine, Room 5108 Gray Building, Medical Center Boulevard, Winston-Salem, NC 27157, USA

Our previous work has shown that high avidity cytotoxic T lymphocytes (CTL) are optimal for virus clearance in vivo and thus it is necessary that an effective vaccine is capable of eliciting high avidity CTL. To determine if vaccination with the paramyxovirus simian virus 5 (SV5) elicits a high avidity response to a model foreign antigen, a recombinant virus was engineered to express chicken ovalbumin (rSV5–Ova). To compare the CTL response elicited with rSV5–Ova and a recombinant vaccinia virus expressing ovalbumin (rVV–Ova), mice were vaccinated intranasally with various doses of each vector and the Ova-specific CTL response was determined by ELISPOT analysis. Here, it has been shown that rSV5 can be equally as effective as rVV in eliciting antigen-specific CTL, in terms of both the total number of CTL and the number of high avidity cells. This has implications for both the design of vaccine vectors and the route utilized for vaccine administration for the elicitation of high avidity CTL responses. The advantages and future potential use of rSV5 vaccine vectors are discussed.

A major goal of vaccine development has been to design viral vectors that elicit cytotoxic T lymphocytes (CTL) that can efficiently clear an ongoing or subsequent infection. One variable that can greatly impact on the efficacy of virus clearance is the functional avidity of a CTL. Our previous results have shown that the avidity of a CTL has profound implications for virus clearance, since the ability of high avidity CTL to recognize lower levels of antigen on target cells allows them to recognize and lyse infected cells at earlier times following infection compared to low avidity cells (Derby et al., 2001). More importantly, a number of adoptive transfer Author for correspondence : Martha Alexander-Miller. Fax j1 336 716 9928. e-mail marthaam!wfubmc.edu

0001-8266 # 2002 SGM

studies have shown that high avidity CTL were capable of reducing the virus burden while low avidity CTL were ineffective (Alexander-Miller et al., 1996 ; Derby et al., 2001 ; Gallimore et al., 1998). These findings support the need for developing vaccination approaches that elicit a strong high avidity CTL response. However, there are major gaps in our understanding of which vectors are best suited for eliciting a potent CTL response containing high avidity CTL. 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 1–4, 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 BBGH

G. D. Parks and M. A. Alexander-Miller

Fig. 1. Ova expression and secretion from rVV–Ova- and rSV5–Ovainfected cells. (A) Structure of rSV5–Ova. The SV5 genome is depicted as a rectangle with vertical bars denoting the intergenic (IG) regions. The 3h leader (le) and 5h trailer (tr) are shown as black boxes. The Ova open reading frame was modified to be flanked on the 3h and 5h sides by the gene end–intergenic–gene start signals from the NP–P/V or the HN–L gene junctions, respectively. (B) Time course of intracellular Ova expression. Monolayers of CV-1 cells were infected at an m.o.i. of 10 with rVV–Ova or rSV5–Ova. At the indicated number of hours p.i., cell lysates were prepared and aliquots analysed by Western blotting using Ova antiserum (top panels) or antisera specific for the SV5 NP and P proteins (rSV5–Ova samples, bottom panel). Note that the amount of lysate analysed for rSV5–Ova-infected cells was twice that of rVV–Ova-infected cells. The two Ova blots were developed simultaneously and had identical exposure times. M, sample from mock-infected cells harvested at 12 h p.i. (C) Ova secretion from virus-infected cells. Monolayers of CV-1 cells infected with rVV–Ova or rSV5–Ova were radiolabelled with Trans[35S] label for 15 min at 15 h p.i. and then incubated in chase medium. At the indicated times, medium was removed and cells were lysed in 1 % SDS. Samples representing 1/8 of the lysed cells and 1/4 of the medium were immunoprecipitated using antiserum specific for chicken ovalbumin before analysis by SDS–PAGE and autoradiography.

of Massachusetts Medical Center) (Craiu et al., 1997) was inserted between the HN and L genes in the rSV5 cDNA clone (Fig. 1A). rSV5–Ova was generated as described previously (He et al., 1997) with minor modifications (Parks et al., 2001). To determine whether inserting the Ova gene into the SV5 genome affected the rate of virus growth, cells were infected at a high m.o.i. with WT rSV5 or rSV5–Ova and a single-step growth analysis was carried out as described previously (Parks et al., 2001). WT and rSV5–Ova viruses showed nearly identical growth rates, as well as final virus titres when measured in CV-1 cells (data not shown). Together, these data BBGI

show that the model CTL antigen Ova can be expressed by rSV5 with no detectable effect on virus growth. To determine how the relative kinetics and level of Ova expression from rSV5 compared with those from rVV, CV-1 cells were infected with rSV5–Ova or rVV–Ova (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 rSV5–Ova was twice that of rVV–Ova. In the case of cells infected with rVV–Ova, 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 rSV5–Ovainfected 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 (" 6–9 h p.i.), consistent with the 5h position of the Ova gene and the 3h to 5h gradient of transcription across the SV5 genome. A pulse–chase 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 rSV5–Ova or rVV–Ova were radiolabelled for 15 min at 15 h p.i. using 100 µCi\ml Trans[$&S] label, washed in PBS and covered with 0n4 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 rSV5–Ova synthesized the same or slightly more Ova during the initial radiolabelling period than did cells infected with rVV–Ova (Fig. 1C, intracellular lanes). The level of intracellular Ova remained relatively constant in cells infected with rVV–Ova 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 rSV5–Ova 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 rSV5–Ova. At later times after infection with rSV5–Ova, the level of Ova was very similar to that found in rVV–Ova-infected cells. Importantly, the noncytopathic nature of rSV5 infection resulted in a continuous

SV5 efficiently elicits high avidity CTL

Fig. 2. rSV5–Ova and VV–Ova induce Ova-specific CTL responses that are quantitatively and qualitatively similar. (A) The number of p.f.u. of virus used for immunization is shown on the x-axis. On day 12 p.i., splenocytes were harvested and analysed using the ELISPOT assay to quantitate the number of Ova-specific IFN-γ-secreting CTL. Cells were cultured in plates previously coated with capture antibody specific for IFN-γ (clone R4-6A2) followed by blocking. After 36 h, plates were washed and incubated with biotinylated anti-IFN-γ antibody (clone XMG1.2) and subsequently with HRP-conjugated avidin. Substrate was added for the detection of IFN-γproducing cells. The number (n) of mice tested for each dose is shown. ND, None detected. (B) Using the optimal doses determined in (A), mice were immunized i.n. with rVV–Ova or rSV5–Ova virus and the number of high and low avidity Ova-specific CTL quantified on day 12 p.i. High avidity CTL are those that produce IFN-γ at the lower concentration of peptide (10−6 M) in the presence of anti-CD8 antibody. Low avidity CTL are those that are CD8-dependent in their response to high peptide concentrations (10−4 M) and are obtained as follows : number of spots following stimulation with 10−4 M in the absence of anti-CD8 antibody minus number of spots following stimulation with 10−4 M in the presence of anti-CD8 antibody. The number of non-specific spots was negligible.

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 rVV–Ova or rSV5–Ova. The number of CTL secreting IFN-γ in response to stimulation with the OVA – peptide was determined by ELISPOT #&( #'%

analysis on day 12 p.i. as previously described (AlexanderMiller, 2000). Fig. 2(A) shows that both viral vectors were capable of inducing an Ova-specific CTL response in mice. The dose of rSV5–Ova that elicited the largest number of CTL was 1i10% p.f.u. On average, this dose of virus elicited 1n6i10% CTL\spleen. For rVV–Ova, in some experiments, the highest number of CTL was elicited by immunization with 1 i 10# 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 anti–Ova CTL (Fig. 2A ; , none detected). Therefore 1i10$ p.f.u. was chosen as the optimal dose. Using this dose, we also detected an average of approximately 1n6i10% Ova-specific CTL\ spleen. Thus, the maximum number of CTL that can be reproducibly elicited by either rSV5–Ova or rVV–Ova is equivalent. Interestingly we found that there was a trend towards decreasing numbers of Ova-specific CTL following immunization with higher doses of rVV–Ova (1i10% and 1i10& p.f.u.) (Fig. 2A and data not shown) and to a lesser extent with rSV5–Ova (1i10& 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. The amount of peptide required for CTL activation\effector function is a reflection of the functional avidity of that cell. High avidity CTL require significantly less peptide when compared with low avidity CTL and in addition are less dependent on CD8 engagement (Alexander et al., 1991 ; Alexander-Miller et al., 1996). Our laboratory has used these differential requirements of high and low avidity CTL to develop a modified ELISPOT assay that allows the enumeration of high and low avidity CTL that are elicited following immunization with virus (Alexander-Miller, 2000). This assay allowed us to compare the relative abilities of rSV5–Ova and rVV–Ova to generate high avidity Ovaspecific CTL. Mice were immunized with the dose of each virus that yielded equivalent numbers of CTL (1i10$ p.f.u. rVV–Ova and 1i10% p.f.u. rSV5–Ova ; 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-γ in the presence of antiCD8 antibody following stimulation with APC pulsed with 10−'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−% M). Fig. 2(B) shows the ratio of low to high avidity CTL for mice vaccinated with rVV–Ova and rSV5–Ova, 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 rVV–Ova was 4n4, while the ratio obtained with rSV5–Ova was 2n8. Although BBGJ

G. D. Parks and M. A. Alexander-Miller

the ratios suggested an increase in the elicitation of high avidity CTL in response to rSV5–Ova versus rVV–Ova, the differences were not statistically significant. Thus, these data showed that immunization with rSV5–Ova results in CTL with similar avidity to those generated in response to rVV–Ova. 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 rSV5–Ova and rVV–Ova 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 i 10$p.f.u. of rVV–Ova or 1i10% p.f.u. of rSV5–Ova, the doses of virus that yielded equivalent numbers of CTL in the acute response. Using the ELISPOT assay, we initially assessed the total number of Ova-specific CTL present in the memory pool (day 40 p.i.). Fig. 3(A) shows the total number of Ovaspecific CTL following immunization with rSV5–Ova or rVV–Ova. The total number of Ova-specific memory CTL generated as a result of immunization with rVV–Ova was 9n3i10$, while 2n8i10$ memory CTL were detected following immunization with rSV5–Ova (Fig. 3A). Thus immunization with rVV–Ova generated approximately 3n3-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 2n2 (2n2i10$ CTL vs 1n0i10$ ; Fig. 3B). Thus, low avidity CTL account for a significant percentage of the increased number of memory CTL generated as a result of rVV–Ova 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 rSV5–Ova is lower compared with rVV–Ova (1n6 vs 3n4). Thus, rSV5–Ova, while eliciting fewer memory CTL overall, appears to be at least as efficient as rVV–Ova in generating high avidity CTL for survival into the memory population. The recent ability to modify genetically paramyxovirus genomes has opened the door to the potential use of these viruses as vaccine delivery vectors. The paramyxovirus SV5 has a number of characteristics that could be exploited to generate a new class of viral vectors including the ability of SV5 to infect non-dividing cells, the apparent lack of packaging constraints, the inability of SV5 to integrate into host DNA and the lack of cytopathic effects in most cell lines tested. 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 (rSV5–Ova) 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 BBHA

Fig. 3. rSV5–Ova is an efficient inducer of high avidity memory CTL. The assay was performed on day 40 or 41 p.i. with rSV5–Ova or rVV–Ova. The number of high and low avidity CTL was determined as in Fig. 2. The total number of Ova-specific CTL (A), the number of high avidity CTL (B), and the ratio of low to high avidity CTL in the long-term memory response (C) are shown.

recombinant vaccinia virus (rVV–Ova) (Figs 2 and 3). Although the total number of CTL observed in the acute response following immunization with rVV–Ova or rSV5–Ova was equivalent, the total number of CTL surviving into the memory pool following administration of rSV5–Ova was somewhat reduced compared with rVV–Ova. 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

SV5 efficiently elicits high avidity CTL

memory response generated following i.p. immunization with rVV–Ova (Alexander-Miller, 2000). In that analysis we found that the ratio of low to high avidity Ova-specific CTL was 2n0 in the acute response compared with 7n1 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 rSV5–Ova or rVV–Ova. 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 rVV–Ova immunization. The reduction in the number of Ovaspecific CTL in our system was also seen in the vaccinia virusspecific 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 (10( p.f.u.) than the highest dose used here for i.n. immunization (10& 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 antigenspecific activity observed in our system with increasing doses of rVV–Ova. In summary, we have found that i.n. immunization with rSV5–Ova is capable of eliciting a robust CTL response that is comparable to that observed with rVV–Ova. Most importantly, vaccination with rSV5–Ova 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. We would like to thank Dr Doug Lyles and Peter Gray for critical reading of this manuscript. This work was supported by grants from the

National Institutes of Health (AI46282 to G. D. P. and AI43591 to M. A. A.).

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Received 6 December 2001 ; Accepted 10 January 2002