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The Journal of Immunology, 2007, 178, 6761-6769
Copyright © 2007 by The American Association of Immunologists, Inc.
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Comparative Prime-Boost Vaccinations Using Semliki Forest Virus, Adenovirus, and ALVAC Vectors Demonstrate Differences in the Generation of a Protective Central Memory CTL Response against the P815 Tumor1

Tanja I. Näslund*, Catherine Uyttenhove{dagger},{ddagger}, Eva K. L. Nordström*, Didier Colau{dagger},{ddagger}, Guy Warnier{dagger},{ddagger}, Mikael Jondal*, Benoît J. Van den Eynde{dagger},{ddagger} and Peter Liljeström2,*

* Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden; {dagger} Ludwig Institute for Cancer Research, Brussels Branch, Brussels, Belgium; and {ddagger} Université catholique de Louvain, Brussels, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tumor-specific Ags are potential target molecules in the therapeutic treatment of cancer. One way to elicit potent immune responses against these Ags is to use recombinant viruses, which activate both the innate and the adaptive arms of the immune system. In this study, we have compared Semliki Forest virus (SFV), adenovirus, and ALVAC (poxvirus) vectors for their capacity to induce CD8+ T cell responses against the P1A tumor Ag and to elicit protection against subsequent challenge injection of P1A-expressing P815 tumor cells in DBA/2 mice. Both homologous and heterologous prime-boost regimens were studied. In most cases, both higher CD8+ T cell responses and better tumor protections were observed in mice immunized with heterologous prime-boost regimens, suggesting that the combination of different viral vectors is beneficial for the induction of an effective immune response. However, homologous immunization with SFV provided potent tumor protection despite a rather moderate primary CD8+ T cell response as compared with mice immunized with recombinant adenovirus. SFV-immunized mice showed a rapid and more extensive expansion of P1A-specific CD8+ T cells in the tumor-draining lymph node after tumor challenge and had a higher frequency of CD62L+ P1A-specific T cells in the blood, spleen, and lymph nodes as compared with adenoimmunized mice. Our results indicate that not only the magnitude but in particular the quality of the CD8+ T cell response correlates with tumor protection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunotherapy for treatment of cancer has been exploited in passive immunotherapy using Abs and specific T cells and in active immunotherapy using tumor cell-based vaccines, proteins, peptides, DNA, recombinant viral particles, cytokines, and dendritic cells (DCs)3 (1, 2, 3). Clinical trials in all the above fields are underway and the results have been reviewed extensively (4, 5, 6, 7, 8, 9, 10) (www.nci.nih.gov and www.wiley.co.uk/genmed/clinical/). Although the most promising results so far have been cancer patients treated with Abs (3) and tumor-infiltrating lymphocytes in combination with lymphodepletion (11), there are several drawbacks with these vaccine regimens since Abs are not very effective against solid tumors, and the generation of tumor-infiltrating lymphocytes from each patient is labor extensive (3, 6).

Several clinical trials using recombinant viral vectors have also been reported. Recombinant viral vectors are attractive vaccine candidates since they have intrinsic properties that activate both the innate and the adaptive arms of the immune system. In particular, recombinant adenovirus and ALVAC (a poxvirus) have been used widely in preclinical and clinical studies (10). By design, these viral vectors are only capable of one round of productive infection because no new viral particles can be produced due to species specificity or as a result of gene deletions (12). Both vaccine vectors have been used in phase I and II clinical trials in patients with carcinomas or adenocarcinomas (10, 13, 14, 15, 16, 17, 18, 19, 20, 21). However, the success has so far been modest. Even though different doses (14, 15) were tested and the vectors were combined with immune stimulatory agents such as IL-2 (14), GM-CSF (16), or B7.1 coexpression (20, 21) or with peptides (17, 18), neither strong T cell responses nor tumor regression were detected in the patients after vaccination. In only a few cases, a partial regression of the tumor burden and stabilization of disease was observed (14, 15, 16, 17, 18, 19, 20, 21). By combining the recombinant viral vectors in heterologous prime-boost regimens, the specific T cell precursor frequencies could be elevated, yet clinical benefits remained elusive (22, 23, 24, 25, 26). Thus, improving immunogenicity of existing viral vectors, testing of new vector combinations, and tailoring the ultimate memory responses are of importance. Vectors that have no or low preexisting immunity in humans are of special interest to avoid antivector effects (14, 16).

For strategies involving recombinant viral vectors, the choice of Ag (Ag-encoding gene) is crucial. Ags that are expressed in tumor cells and not in healthy cells represent important targets because possible autoimmune side effects can be avoided. Shared tumor-specific Ags encoded by cancer-germline genes are expressed on various types of human tumors, suggesting that several groups of cancer patients might benefit from vaccines targeting these Ags. These genes are also normally expressed in male germline (27, 28), but because these cells do not express HLA molecules they cannot present the antigenic peptide to CTLs. The first human shared tumor-specific Ags discovered were encoded by the MAGE genes (29, 30, 31) and later the BAGE, GAGE, LAGE, and NY-ESO-1 families were described (1). These Ags are expressed in melanoma cells, as well as in a variety of adenocarcinomas (32), carcinomas, and sarcomas (27). Like the human MAGE Ags, the murine tumor Ag P815A, encoded by gene P1A, is expressed only in testis and placenta (33, 34). The mastocytoma P815 expresses five known tumor Ags (A, B, C, D, and E) and was established in DBA/2 mice by treatment with the tumor inducing agent methylcholanthrene (35, 36). The P815 tumor mouse model is a relevant model to test different vaccination modalities with possible applications for human shared tumor-specific Ags.

Recombinant adenovirus, ALVAC, and Semliki Forest virus (SFV) have been tested in several preclinical studies (10, 37, 38, 39, 40, 41, 42, 43). It has been shown previously that SFV viral particles can be used for multiple immunizations without hampering the additive effect on the immune response, making the SFV viral vector an attractive vector (38, 42). Adeno- and SFV viral particles expressing the P1A Ag have been shown previously to induce P1A-specific cytotoxic T cell responses in blood (12) and in spleen (44, 45). The SFV-P1A-immunized mice were also partially protected against a P815 tumor challenge (44, 45). In contrast, ALVAC viral particles expressing the P1A Ag have not been used in any preclinical studies to our knowledge. Similar to results from clinical trials, mice immunized with recombinant viral vectors in heterologous prime-boost regimens in preclinical tumor models showed increased frequencies of specific CD8+ T cells compared with mice immunized twice with the same vector (10, 46, 47, 48, 49, 50, 51, 52, 53, 54). Heterologous prime-boost-immunized mice also showed prolonged survival (55, 56).

In this study, we have used the P815 tumor mouse model to compare the viral vectors SFV, adenovirus, and ALVAC side by side for their ability to elicit P1A-specific CD8+ T cell responses and their capacity to induce protection against P815 tumor challenge. The viral vectors were tested in both homologous and heterologous prime-boost regimens because many studies have shown that increased T cell responses can be achieved by combining different viral vectors (46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61). Our results demonstrate both quantitative and qualitative differences in the CD8+ T cell response generated by the different viral vectors and show that tumor specific central memory CD8+ T cells are of importance for protection against tumor challenge.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and immunizations

DBA/2 Ola Hsd-inbred female mice were obtained from Harlan and kept at animal houses at Karolinska Institutet and Ludwig Institute for Cancer Research in pathogen-free environment. The mice were between 8 and 14 wk old when they were immunized in homologous or heterologous prime-boost regimens 2 wk apart. Adeno-P1A truncated (P1At) virus (108 PFU), SFV-P1A (107 IU), SFV-P1At (107 IU), and SFV-LacZ virus (107 IU) were given intradermally (i.d.) in 100 µl of PBS divided between both ears. The ALVAC-P1At virus (107 PFU) was given i.v. in 100 µl of PBS, and the L1210.P1A.B7.1 cells (106 living cells) were injected i.p. in 200 µl of PBS. All animal care and treatment was in accordance with standards approved by the local ethics committee.

Viruses

The recombinant adenovirus encoding the first 83 residues of the P1A protein (adeno-P1At) was constructed as described previously (12). Recombinant ALVAC encoding the first 83 residues of the P1A protein (ALVAC-P1At) was obtained from J. Tartaglia and J. Tine (Virogenetics Troy, NY). The SFV two-helper RNA system has been described previously (62). Briefly, the P1A gene was amplified by PCR from the pSFV1-P1A vector with XmaI overhang primers (5'-CACGACCCGGGATGTCTGATAACAAGAAACCA-3' and 5'-AATTACCCGGGCTAAGGTGAGAAGC-3'). The P1At gene was amplified from pcD-Sr{alpha}-P1At by PCR using XmaI overhang primers (5'-AATACCCGGGATGTCTGATAACAA-3' and 5'-AATACCCGGGCTAATCGACAGA-3'). The P1A and P1At PCR products were cloned into pSFVb12A. The virus stocks were titrated as previously described (62) by using anti-P1A Abs and anti-Replicase Abs. SFV-LacZ has been described previously (63).

Cell lines

Different P815 subclones were used in this study: P511 and P1.HTR3 (64) both expressing the P1A Ag and P1.204, which has lost the P1A expression (35). L1210.P1A.B7.1, a DBA/2 leukemia cell line, has been described previously (65). The P815 and L1210 cell lines and BHK cells were grown as described previously (33, 62).

Mixed lymphocyte tumor culture (MLTC)

Blood was collected 2 wk after the last immunization, and the PBLs were enriched by Ficoll-Paque PLUS gradient (Amersham Biosciences). The lymphocytes were stimulated with 1.5 x 105 irradiated (10,000 rad) L1210.P1A.B7.1 cells and 2 x 106 irradiated (3,000 rad) syngeneic spleen cells in 48-well plates in MTLC medium as described previously (33). Lytic activity was measured 7 days later in a chromium release assay.

Chromium release assay

The in vitro-restimulated lymphocytes were collected and added to V-shaped 96-well plates and diluted 3-fold as described previously (33). 51Cr-labeled P511 or P1.204 (1000 cells) and 1 x 105 unlabeled P1.204 competition cells were added in a final volume of 150 µl of DMEM 5% FCS. Radioactivity was measured after 4 h of incubation at 37°C from supernatant (100 µl) mixed with 150 µl of scintillation fluid and read in PerkinElmer gamma counter or with Wizard 1470 Automatic gamma counter (Wallac), where the supernatant was measured directly. The lytic activity was expressed in LUs, the definition of 1 LU is the number of lymphocytes that lyse 50% of 104 target cells in 4 h. The LUs were estimated from the specific release obtained at three different E:T ratios chosen in the linear range of the lysis curve by means of regression (1 – e–kx) and expressed as LU/106 effector cells. Specific LUs above 0.1 were regarded as a positive response. Statistical analysis was performed using two-tailed Mann-Whitney U test.

Tumor challenge

Three weeks after PBL collection, the mice were challenged s.c. in the flank with 106 P1.HTR3 cells in 100 µl of PBS. The tumor volume was measured twice per week using a caliMax device (Fine Science Tools), and mice were euthanatized when the tumor volume reached 1.5 cm3. When the last naive mouse was euthanatized, the remaining mice were monitored for tumors at least for an additional 30 days. Mice that had to be euthanized due to outgrowth of tumor Ag loss variants were included in the groups of mice that did not regress the tumor.

FACS analysis

Blood and half of the splenocytes from single-cell suspensions were enriched in lymphocytes by Ficoll gradient. The inguinal tumor draining lymph node (LN) was disrupted in medium, and lymphocytes were collected by centrifugation. All lymphocytes from the LN and PBLs and 3 x 106 lymphocytes from the spleen were stained for flow cytometry. Cells were stained for 15 min at room temperature in PBS buffer containing 1% BSA, 0.1 µM PE-labeled H-2Ld/P1A tetramer, and CD16/32-FITC (1/50) (BD Pharmingen). Then, anti-CD8{alpha}-PerCp (1/100), anti-CD4-FITC (1/50), anti-CD11b-FITC (1/100), anti-CD19-FITC (1/50), and anti-CD62L-allophycocyanin-labeled Abs (all from BD Pharmingen) were added, and cells were incubated for 15 additional min at room temperature. The cells were washed in PBS-1% BSA, pelleted by centrifugation at 500 x g, and resuspended in PBS-1% BSA for flow cytometry analysis. CD8+ FITC-negative cells (104) were collected for calculation of the numbers of P1A-H2Ld tetramer-positive cells. CD8+ P1A-H2Ld tetramer-positive cells were collected for CD62L analysis.

IFN-{gamma} ELISPOT

IFN-{gamma} ELISPOT analysis was performed on freshly isolated splenocytes as described previously (38). Spleen cells (2 x 105) from individual mice were stimulated with medium alone, 2 µg/ml Con A (Sigma-Aldrich), or 2 µg/ml P1A H-2Ld peptide LPYLGWLVF. The spots were counted using an ELISPOT reader (Axioplan 2 Imaging; Zeiss) and expressed as number of spots per 106 splenocytes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD8+ T cell responses after prime-boost immunization

To compare the different viral vectors for their capacity to induce P1A-specific CD8+ T cell responses, we first optimized the dose (mice were immunized with doses from 107 to 109 PFU for adeno-P1At, 106 to 108 PFU for ALVAC-P1At, and 106 and 107 IU for SFV-P1A viral vectors) and immunization route (i.d., i.v., s.c., i.p.) by measuring the induction of a P1A-specific CD8+ T cell response in PBLs (data not shown). The dose and route that generated the highest P1A-specific CD8+ T cell response for each viral vector was then used in subsequent homologous and heterologous prime-boost regimens. As a positive control, we used the leukemia cell line L1210.P1A.B7.1, which has been shown to induce very strong P1A-specific cytotoxic activity and protection against P1A-expressing tumor challenge (66). Following administration of the different viral vectors, PBLs were collected 14 days after the last immunization, and the CTL activity was measured after 7 days of in vitro restimulation. By assessment of the CTL response in PBLs instead of spleen, we could correlate the CTL response to the tumor protection in each individual mouse. All immunized mice showed a significant cytotoxic activity compared with the nonimmunized mice (p < 0.001) (Fig. 1A and Table I). Among the mice immunized twice with the same viral vector, the highest lytic activity was observed in the adeno-P1At group while significantly lower cytotoxic activity was found in SFV-P1A-immunized mice (p < 0.01). The lowest cytotoxic activity was detected in ALVAC-P1At-immunized mice as compared with adeno-P1At (p < 0.001) and SFV-P1A-immunized mice (p < 0.01) (Fig. 1A and Table I).


Figure 1
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  		FIGURE 1. P1A-specific CD8+ T cell responses induced in mice immunized by homologous and heterologous prime-boost regimens. A, P1A-specific lytic activity in PBL after 7 days in vitro restimulation with L1210.P1A.B7.1 cells. The cytotoxic activity is expressed as specific LUs per million effector cells. The dots represent the lytic activity generated in individual mice, and the group median values are indicated by bars. A value of specific LUs >0.1 is regarded as a positive cytotoxic response. Data are pooled from several experiments with 9–11 mice/group. All groups of mice were tested for statistical significance. B, Lytic activity induced in mice immunized twice with SFV-P1A or SFV-P1At viral particles.

 

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  		Table I. Summary of data generated from CTL analysis and tumor protection

 
In general, mice immunized with heterologous prime-boost regimens displayed a lytic activity that was significantly higher than mice immunized twice with the same vector (p < 0.01). The only exception was mice primed with ALVAC-P1At, followed by an SFV-P1A boost, which did not differ significantly from mice either immunized with ALVAC-P1At or SFV-P1A alone. It was clear that, in this model, the viral vectors differed in their priming ability. ALVAC-P1At appeared to be somewhat weaker for priming than adeno-P1At and SFV-P1A, irrespective of the vector used for the boost (Fig. 1A and Table I). However, as a boosting vector, ALVAC-P1At was similar to the other two viral vectors. The highest cytotoxic activities were found in mice primed with SFV-P1A, suggesting that the SFV vector is good as a prime. Whether the adeno-P1At vector worked better as prime or boost was dependent on which vector it was combined with. It worked significantly better as a prime when combined with ALVAC-P1At (p < 0.01), but the reverse was observed when it was combined with SFV-P1A (p < 0.05) (Fig. 1A and Table I).

In our original experiments, for historical reasons, the adeno-P1At and the ALVAC-P1At viral vectors encoded the first 83 residues of the P1A protein (P1At), including the known antigenic peptide, which is presented to CD8+ T cells by H-2Ld molecules. In contrast, the recombinant SFV encoded the full-length P1A protein. Therefore, we wanted to ensure that the observed difference between the vectors was not linked to the difference in length of the inserted P1A gene. Thus, a SFV vaccine vector carrying the truncated version of the P1A gene (SFV-P1At) was made and compared with the SFV-P1A vector. A similar lytic activity was induced in the groups immunized with SFV-P1At and SFV-P1A (Fig. 1B), indicating that the length of the inserted P1A gene did not affect immunogenicity.

Tumor protection after prime-boost immunization

To study whether the various vaccination strategies could result in protection against a tumor challenge, immunized mice were challenged by s.c. injection of the P815 subline P1.HTR3 cells 5 wk after the last immunization (i.e., 3 wk after PBL collection). The tumor volume was then measured twice a week. All mice had palpable tumors by day three after the challenge, demonstrating the aggressive nature of this tumor. Ten days after the tumor challenge, the tumors began to regress in some mice and eventually became nonpalpable. Most of these mice then remained tumor free throughout the experiments. However, in some rare occasions the tumor reappeared, probably caused by outgrowth of tumor-Ag loss variants (35), and these mice eventually had to be euthanatized due to a high tumor burden. These mice were included in the group that did not regress the tumor. In mice immunized twice with the same vector the highest survival scores (60–90%, depending on experiment) were observed in SFV-P1At and SFV-P1A-immunized mice (Table I). Survival rates in these groups were comparable to that of the positive control group, L1210.P1A.B7.1, despite the induction of significantly lower CTL responses. Hence, neither the lytic activity nor the survival was dependent on the length of the P1A gene (Fig. 1B and Table I). About half (50–70%) of the mice immunized twice with adeno-P1At were protected against the tumor challenge (Table I), whereas mice immunized twice with ALVAC-P1At had the lowest survival rate (30–44%) among all the vaccinated groups tested (Table I).

Most of the heterologous-immunized groups showed a survival rate of at least 50%. Mice primed with SFV-P1A and boosted with adeno-P1At showed exceptional surviving rates (90–100%) (Table I). Heterologous prime-boost almost invariably increased the survival rate for ALVAC-P1At-primed mice when adeno-P1At or SFV-P1A was used as boost compared with mice immunized with ALVAC-P1At twice. However, the higher lytic activity observed after heterologous boost did not correlate with better tumor protection. This was clear in the group primed with adeno-P1At and boosted with ALVAC-P1At (median LU = 36.2), which had similar survival rate to mice immunized twice with adeno-P1At (median LU = 3.1) or primed with ALVAC-P1At and boosted with adeno-P1At (median LU = 10.9) (Table I and Fig. 2). This was also observed in mice primed with SFV-P1A and boosted with ALVAC-P1At (median LU = 52.0), where tumor protection was lower than that of mice immunized twice with SFV-P1A (median LU = 2.3). In the nonimmunized mice, there was always one mouse in each experiment that survived the tumor challenge (Table I). Collectively, the tumor protection did not follow the level of P1A-specific CD8+ T cell response. The lytic activity and the survival rates were clearly discordant in mice immunized twice with the same vector (Fig. 2). Exclusion of the mice that had to be euthanized due to regrowth of Ag loss tumors did not alter the mean protection significantly in any of the groups of mice (data not shown).


Figure 2
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  		FIGURE 2. Relationship between LUs and survival (%). The filled symbols represent mice immunized with the same vector twice, and the open symbols represent mice immunized in heterologous prime-boost regimens. The y-axis represents LUs, and the x-axis represents survival (%). Data are derived from Table I.

 
Number of P1A-specific CD8+ T cells in SFV-P1A- and adeno-P1At-immunized mice

Because the level of cytolytic activity detected in the blood could not fully explain the high survival rate in mice immunized twice with SFV-P1A viral particles as compared with the adeno-P1At-immunized group, we asked whether other parameters could better explain survival outcome. Such could be the number and/or tissue distribution of the P1A-specific CD8+ T cells. To address these questions, we used tetramers of H-2Ld molecules folded with the P1A peptide to measure the number of P1A-specific CD8+ T cells. Cells from spleen and blood were analyzed 5 wk after the last immunization with SFV-P1A and adeno-P1At vectors or L1210.P1A.B7.1 cells, a time point corresponding to the tumor challenge in the previous experiments. We found that the number of P1A-specific CD8+ T cells in blood and spleen were significantly lower in the SFV-P1A-immunized mice than in adeno-P1At- and L1210.P1A.B7.1-immunized mice (Fig. 3, A and B). This was also confirmed by ELISPOT, where lower numbers of IFN-{gamma}-producing cells were detected in spleen in the SFV-P1A-immunized mice compared with adeno-P1At-immunized mice (Fig. 3C). The same trend was observed in pooled blood and LNs (data not shown). To verify these results, mice were also analyzed with P1A tetramers 10 days and 6 wk after the last immunization with the same outcome (data not shown). Thus, in agreement with the CTL data, the higher tumor protection in the SFV-P1A-immunized mice was not due to a larger pool of P1A-specific CD8+ T cells.


Figure 3
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  		FIGURE 3. Numbers of P1A-specific CD8+ T cells in blood and spleen in adeno-P1At-, SFV-P1A-, and L1210.P1A.B7.1-immunized mice. The numbers of the P1A-specific CD8+ T cells were determined with H-2Ld/P1A tetramers 5 wk after the last immunization in mice immunized twice with the same vector in PBL (n = 3, pooled before FACS analysis) (A) and spleen (n = 3) (B). The experiment was repeated three times. Representative data are shown. C, IFN-{gamma} ELISPOT on splenocytes from SFV-P1A and adeno-P1At homologous-immunized mice. The response was analyzed 5 wk after the last immunization and is presented as numbers of IFN-{gamma}-producing cells per 106 splenocytes. Each dot represents an individual mouse. •, IFN-{gamma} secretion from P1A-peptide stimulated splenocytes; {circ}, medium controls. Bars indicate median values.

 
Expansion of P1A-specific CD8+ T cells posttumor challenge

Although the total amount of P1A-specific CD8+ T cells was lower in SFV-P1A-immunized mice as compared with adeno-P1At-immunized mice, we considered whether the better protection detected in the SFV-P1A-immunized animals could be due to a more rapid expansion of the vaccine-induced P1A-specific CD8+ T cells after challenge with P1.HTR3 tumor cells. By palpation of the tumors postchallenge, we noted a decrease in tumor volume around day 10 postchallenge in the mice that eventually were capable of rejecting the tumor. To follow the expansion of the P1A-specific CD8+ T cells at the time of tumor regression, we immunized mice twice with adeno-P1At, SFV-P1A, or L1210.P1A.B7.1 cells and challenged them with P1.HTR3 cells 5 wk after the last immunization. At 4, 10, 11, and 15 days after tumor challenge, cells from blood, spleen, and the inguinal tumor draining LN were analyzed with P1A tetramers. The expansion of the P1A-specific CD8+ T cells was estimated by comparing their number before and after the tumor challenge. However, in the L1210.P1A.B7.1-immunized mice, the expansion in the inguinal tumor draining LN was compared with the level at 4 days postchallenge.

In PBLs from adeno-P1At-immunized mice, we detected a small increase in P1A-specific CD8+ T cells 4 days after the tumor challenge, which were still slowly expanding 15 days postchallenge (Fig. 4A). In the spleen and the inguinal tumor draining LN, we could only detect an increase of the P1A-specific CD8+ T cells 10 and 11 days postchallenge, respectively. Fifteen days after tumor challenge, the levels had contracted to prechallenge levels in both tissues (Fig. 4, B and C). In contrast to the adeno-P1At-immunized mice, the SFV-P1A-immunized mice showed a larger expansion of the P1A-specific CD8+ T cells peaking at 11 days after the tumor challenge in all the tissues tested. Thereafter, the P1A-specific CD8+ T cell numbers contracted but to frequencies still above prechallenge levels (Fig. 4). Interestingly, while the total number of P1A-specific CD8+ T cells remained similar in PBLs and spleen in the adeno-P1At- and SFV-P1A-immunized mice, the SFV-P1A-immunized mice had a very large expansion of the P1A-specific CD8+ T cells in the inguinal tumor draining LN. The L1210.P1A.B7.1-immunized mice had a fast expansion of the P1A-specific CD8+ T cells that was detected already 4 days after the tumor challenge, although the final numbers of P1A-specific T cells were not significantly higher than for SFV-P1A- and adeno-P1At-immunized mice. The numbers of P1A-specific CD8+ T cells in the L1210.P1A.B7.1-immunized mice thereafter declined slightly and reached a steady state in both blood and spleen (Fig. 4, A and B). In the inguinal tumor draining LN, we could detect a slow but steady increase of the P1A-specific CD8+ T cells over time in the L1210.P1A.B7.1-immunized mice, even at 15 days postchallenge (Fig. 4C). In the nonimmunized mice, we detected an expansion of the P1A-specific CD8+ T cells 4 days after the tumor challenge with levels increasing until day 11; thereafter, the levels decreased in all tissues tested (Fig. 4).


Figure 4
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  		FIGURE 4. Kinetics of the P1A-specific CD8+ T cell response in adeno-P1At-, SFV-P1A-, and L1210.P1A.B7.1-immunized mice. Mice were immunized twice with the same vector and challenged 5 wk after the last immunization. At different time points postchallenge (4, 10, 11, and 15 days), cells from PBLs (n = 3–8) (A), spleen (n = 3–8) (B), and the inguinal tumor draining LNs (n = 3–6, pooled at days 4 and 11) (C) were analyzed for P1A-specific CD8+ T cells by FACS analysis using H-2Ld/P1A tetramers. Fold expansion of P1A-specific CD8+ T cells are indicated above the bars. n.d. = not determined.

 
CD62L expression on P1A-specific CD8+ T cells

It has previously been shown that there are differences in proliferation capacity between the two T cell memory populations, where central memory T cells (TCM) have a better capacity to expand as compared with effector memory T cells (TEM) (67, 68, 69). To determine whether the difference detected in the expansion of the P1A-specific CD8+ T cells correlated with different types of memory T cells, cells from blood, spleen, and LNs were analyzed for CD62L expression on P1A-specific CD8+ T cells in SFV-P1A- or adeno-P1At-immunized mice 5 wk after the last immunization. CD62L is a commonly used marker distinguishing different memory T cell populations because it is expressed on TCM but not on TEM (70). As before (Fig. 3, A and B), the total number of P1A-specific T cells was higher in the adeno-P1At-immunized mice compared with the SFV-P1A group (data not shown). However, a higher frequency of CD62L-positive cells was detected in the P1A-specific CD8+ T cell population in the SFV-P1A-immunized mice compared with the adeno-P1At group in all tissues tested (blood (p < 0.05), spleen (p < 0.001), and LNs (p < 0.001)) (Fig. 5).


Figure 5
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  		FIGURE 5. CD62L expression in the P1A-specific CD8+ T cell population. Mice were immunized twice with SFV-P1A or adeno-P1At viral particles. Five weeks after the last immunization, blood, spleen, and LNs were analyzed using flow cytometry for CD62L expression on the P1A-specific CD8+ T cells (n = 8/group, spleen and LNs were analyzed as individual mice; the PBL samples were pools from two mice). The data generated from the LNs need to be taken with caution due to low numbers of P1A-specific CD8+ T cells in this tissue compartment. Mean ± SD are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It is well documented that rejection of P815 tumors is CD8+ T cell dependent and that the major target for tumor rejection is the P1A CTL epitope (66). In this study, we confirm the importance of the P1A Ag for rejection of P1A-expressing tumors because, for example, SFV-P1A immunization resulted in good survival from P1.HTR3 tumor challenge. Moreover, SFV-LacZ-immunized mice did not differ from nonimmunized mice in tumor progression, suggesting that the tumor regression required a P1A-specific immune response (data not shown). Although the protective role of P1A-specific CD8+ T cells is clear, the contribution of the CD4+ T cells in this tumor model has not been fully evaluated. CD4+ T cells might help to further modulate the CD8+ T cell response because the P1At Ag in this study does contain a MHC class II 12-mer epitope, part of which is overlapping the P1A CTL epitope (71). However, the SFV-P1A and SFV-P1At viral particles induced similar cytotoxic levels and survival frequencies, suggesting that no additional MHC class II epitopes of major importance for the induction of the P1A-specific CD8+ T cells and survival exists in the deleted part of the P1At protein. NK cells, which potentially could participate in the defense against tumors, do not play a role in the P815 tumor regression because these tumor cells are resistant to NK-mediated cell lysis (72).

In this study, we have compared the viral vectors SFV, adenovirus, and ALVAC expressing the tumor Ag P1A for their ability to induce P1A-specific CD8+ T cells and protection against P815 tumor challenge in mice. The P1A-specific CD8+ T cell levels and survival after tumor challenge were evaluated after homologous as well as after heterologous prime-boost regimens. We could show that heterologous prime-boost induced higher specific CD8+ T cell responses, which is in agreement with other vaccine studies in disease models such as HIV (73) and malaria (60, 61). Elevated immune responses after heterologous prime-boost might partly depend on avoidance of boosting antivector responses and focusing the immune responses toward amplification of the Ag-specific T cells (55). However, the contribution to the T cell responses by the individual viral vectors in the heterologous prime-boost-immunized mice was not addressed in this study and should be assessed after a single prime with the individual vaccine vectors. The heterologous prime-boost-immunized mice were also better protected against a tumor challenge in the majority of combinations tested. The highest cytotoxic activity and survival rates (90–100%) were detected in mice immunized with SFV-P1A prime and adeno-P1At boost. It was clear in this study that some viral vectors worked better as prime (SFV-P1A) or boost (ALVAC-P1At), although this varied according to the vector they were combined with (adeno-P1At). However, the optimal regimen (vector, dose, and route) might very well be different in other tumor or disease models.

We also observed that a high lytic activity does not necessarily correlate with a good protection against a tumor challenge, at least in this model. In previous studies, both an association (74, 75, 76) and lack of association (77, 78) between a specific T cell response and tumor regression have been reported. The latter is in accordance with our findings, especially in the mice immunized twice with the same vector. SFV-P1A-immunized mice were highly protected against a tumor challenge despite only moderate induction of P1A-specific cytotoxic activity. The reverse was found for adeno-P1At-immunized mice. The increased survival of the SFV-P1A-immunized mice compared with adeno-P1At-immunized mice did not depend on a larger pool of P1A-specific CD8+ T cells in the blood or spleen at the time of tumor challenge, as shown both by H-2Ld/P1A tetramer staining and IFN-{gamma} ELISPOT. Instead the increased survival of the SFV-P1A-immunized mice was associated with rapid expansion of the P1A-specific CD8+ T cells after tumor challenge, especially in the inguinal tumor draining LN. However, the total numbers of P1A-specific CD8+ T cells in blood and spleen reached similar levels in the SFV-P1A- and the adeno-P1At-immunized mice after tumor challenge, suggesting that it was not the level of P1A-specific CD8+ T cells per se that was important, but rather the type or quality of the activated population. Hence, the nature of the memory population formed by the different viral vectors seemed to be of importance for tumor regression.

Memory T cells can be divided into TCM and TEM by surface markers, such as CD62L and CCR7, and by function, such as direct cytolytic activity and IL-2 secretion. CD62L, a LN homing receptor, is expressed on TCM but not on TEM. The preferred homing of TCM to LNs has been suggested to generate more effective interaction between TCM and APCs. It has been shown that TCM have a higher proliferation capacity after Ag encounter, and it has also been suggested that TCM have reduced direct cytolytic activity compared with TEM (79). Clearly, there are differences in function between the different memory populations, but whether TCM or TEM are more beneficial seems to vary between different disease models. In some viral infections, a dominant TEM population is beneficial for viral clearance while others depended on a TCM response (80). For protection against tumors, it was shown recently that TCM confer better antitumor immunity compared with TEM (68), and it has been suggested that the optimal cancer immune therapy should strive to generate strong TCM responses (81). Our finding support this view because SFV-P1A-immunized mice, with a high ratio of P1A+CD62L+/P1A+CD62L–CD8+ T cells, were highly protected against a tumor challenge compared with adeno-P1At-immunized mice. The reason for the differences in induction of memory T cell populations by the two viral vectors, SFV and adenovirus, could be due to a difference in the way they activate APCs. It has been suggested recently that primarily TCM are generated when there is a high T cell/APC ratio and the contrary for generation of TEM (82). Why different numbers of activated APCs are induced might be due to different amount of P1A Ag produced by the vectors or by differences in the strength of the innate signals induced by the two viral vectors (83). Recent results from our laboratory have indeed suggested that several innate pathways are activated during SFV replication. Strong type I IFN responses are induced by SFV vectors, and they can act as adjuvant to coadministered protein on Ab production (84, 85). We have also recently shown that the stimulatory effects by the SFV vectors on the innate immune system act in part through the dsRNA receptors TLR3 and Mda5 (86, 87).

The question is whether the difference detected in the protection against tumor challenge in this study can be explained only by the different ratio of TCM and TEM cell populations in SFV-P1A and adeno-P1At-immunized mice. The total number of P1A-specific CD8+ T cells was higher in the adeno-P1At-immunized mice, which implies that the actual number of TCM can be equal or even higher compared with the SFV-P1A-immunized mice. However, the SFV-P1A-immunized mice were more protected against a tumor challenge than the adeno-P1At-immunized mice, suggesting that the ratio of TCM and TEM is more important than the total number of TCM. This might be due to competition for H-2 molecules between the two memory populations. This phenomenon has been reported to occur between naive and memory T cells (88), as well as in the development of TCM and TEM populations (82). This might even be more pronounced in the P815 tumor model because H-2Ld molecules are expressed at quite low levels (89).

Other parameters (79, 90, 91, 92, 93, 94), such as the activation status of the responder APCs, the cytokine patterns, and other surface markers on the P1A-specific CD8+ T cells, have not been investigated in this study but might also play a role as well in explaining the more efficient regression of P815 tumors in the SFV-P1A-immunized mice. Further in-depth characterization of the quantitative and qualitative T cell responses induced by the different viral particles and their correlation to memory T cell responses and tumor protection needs to be performed to clarify these issues.


   Acknowledgments
 
We thank Dominique Donckers for excellent technical assistance (Ludwig Institute for Cancer Research, Brussels, Belgium) and the staff in the animal house at Department of Microbiology, Tumor, and Cell Biology, Karolinska Institutet. We also thank J. Tartaglia and J. Tine, Virogenetics, for the ALVAC-P1At viral construct.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Swedish Research Council, the European Union 5th Framework Program, the European Community (QLGA-CT-2000-60013), and the Fondation contre le Cancer. Back

2 Address correspondence and reprint requests to Dr. Peter Liljeström, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Nobels väg 16, Box 280, 17177 Stockholm, Sweden. E-mail address: Peter.Liljestrom{at}ki.se Back

3 Abbreviations used in this paper: DC, dendritic cell; LN, lymph node; MLTC, mixed lymphocyte tumor culture; P1At, P1A truncated; SFV, Semliki Forest virus; TCM, central memory T cell; TEM, effector memory T cell. Back

Received for publication November 22, 2006. Accepted for publication March 13, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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