Characterization of -Specific Immune Responses Induced by

This information is current as Rinke Bos, Suzanne van Duikeren, Thorbald van Hall, of September 27, 2021. Marjolein M. Lauwen, Mark Parrington, Neil L. Berinstein, Bryan McNeil, Cornelis J. M. Melief, J. Sjef Verbeek, Sjoerd H. van der Burg and Rienk Offringa J Immunol 2007; 179:6115-6122; ;

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Characterization of Antigen-Specific Immune Responses Induced by Canarypox Virus Vaccines1

Rinke Bos,* Suzanne van Duikeren,* Thorbald van Hall,* Marjolein M. Lauwen,* Mark Parrington,‡ Neil L. Berinstein,‡ Bryan McNeil,‡ Cornelis J. M. Melief,* J. Sjef Verbeek,† Sjoerd H. van der Burg,* and Rienk Offringa2*

Avipoxvirus-based vectors, such as recombinant canarypox virus ALVAC, are studied extensively as delivery vehicles for vaccines against and infectious diseases. Effective use of such vaccines is expected to benefit from proper understanding of the interaction between these viral vectors and the host . We performed preclinical vaccination experiments in a murine tumor model to analyze the immunogenic properties of an ALVAC-based against carcinoembryonic Ag (ALVAC- CEA), a tumor-associated autoantigen commonly overexpressed in colorectal . The protective CEA-specific immunity induced by this vaccine consisted of CD4؉ T cell responses with a mixed Th1/Th2 cytokine profile that were accompanied by Downloaded from -potent humoral responses, but not by CEA-specific CD8؉ CTL immunity. In contrast, protective immunity induced by a CEA specific DNA vaccine (DNA-CEA) consisted of Th1 and CTL responses. Modification of the ALVAC-CEA vaccine through coinjection of DNA-CEA, admixture with CpG oligodeoxynucleotides, or supplementation with additional transgenes encoding a triad of costimulatory molecules (TRICOM) did not result in induction of CEA-specific CTL responses. Even though these results suggested that ALVAC does not elicit Ag-specific CTLs, immunization with ALVAC vaccines against other Ags efficiently induced http://www.jimmunol.org/ CTL responses. Our data show that the capacity of ALVAC vaccines to elicit CTL immunity against transgene-encoded Ags critically depends on the presence of highly immunogenic CTL epitopes in these Ags. This consideration needs to be taken into account with respect to the design and evaluation of vaccination strategies that use ALVAC-based vaccine. The Journal of Immunology, 2007, 179: 6115–6122.

ecombinant avipoxviruses, including ALVAC (canary- C57BL/6 mice induced protective immunity against an otherwise pox virus), are used as vehicles for the development of lethal dose of CEA-overexpressing tumor cells, MC38-CEA. Even R vaccination strategies against cancer, malaria, tuberculo- though MC38-CEA tumor cells express MHC class I, but not sis and AIDS. Their inability to replicate in other than birds MHC class II, protective immunity was found to depend on the by guest on September 27, 2021 provides an excellent safety barrier against vaccine-associated CEA-specific CD4ϩ Th response. CD8ϩ CTL were found to be complications in humans. Despite this abortive infection, the Ags dispensable (6). The lack of involvement of CEA-specific CTL in encoded by the foreign genes inserted into the ALVAC vector are tumor eradication prompted the question concerning which CD4ϩ expressed and become presented to the host immune system (1, 2). T cell-dependent effector mechanisms would mediate tumor erad- One of the potential applications of ALVAC concerns therapeutic ication in this setting and, furthermore, whether ALVAC-CEA im- vaccination against through induction of immune munization of C57BL/6 mice would be capable of inducing CEA- responses against carcinoembryonic Ag (CEA),3 a tumor-associ- specific CTLs at all. ated autoantigen that is overexpressed by several cancer types of Our present study revealed that ALVAC-CEA immunization in epithelial origin (3). The capacity of poxvirus-based vaccines to this experimental setting elicited a CEA-specific CD4ϩ T cell re- elicit CEA-specific antitumor immunity is studied extensively in sponse displaying a mixed Th1/Th2 profile that was accompanied both preclinical murine tumor models and cancer patients (4, 5). by a potent CEA-specific humoral response, but not by any mea- We have previously reported that ALVAC-CEA immunization of surable CEA-specific CTL immunity. Accordingly, tumor clear- ance involved Ab-dependent effector mechanisms. Although the type of immunity elicited by ALVAC-CEA was protective in this *Department of Immunohematology and Blood Transfusion, Tumor Immunology mouse tumor model, it may not be optimal for antitumor activity Group, †Department of Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands; and ‡Sanofi Pasteur, Toronto, Ontario, Canada in patients with colorectal cancer, in particular because one would Received for publication July 19, 2007. Accepted for publication August 27, 2007. expect anti-CEA Abs to be distracted from targeting the tumor due to the elevated levels of CEA in the circulation of these patients 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 (3). We therefore investigated why ALVAC-CEA failed to induce with 18 U.S.C. Section 1734 solely to indicate this fact. a CEA-specific Th1/CTL response and tested whether modifica- 1 This work was supported by Sanofi Pasteur Toronto and by Grant UL 2000-2035 tions would empower this vaccine to elicit CEA-specific CTL from the Dutch Cancer Society. immunity. 2 Address correspondence and reprint requests to Dr. Rienk Offringa, Department of Immunohematology and Blood Transfusion, Tumor Immunology Group, E3-Q, Lei- den University Medical Center, Albinusdreef 2, 2333 RA Leiden, The Netherlands. Materials and Methods E-mail address: [email protected] Mice 3 Abbreviations used in this paper: CEA, carcinoembryonic Ag; HPV, human papil- b lomavirus; ADCC, Ab-dependent cellular cytotoxicity; ODN, oligodeoxynucleotide. C57BL/6 Kh (B6, H-2 ) were bred in our Leiden University Medical Cen- ter facilities (Leiden, The Netherlands). Fc receptor ␥-chain knockout mice Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 and C1q knockout mice were provided by J. S. Verbeek (Leiden University www.jimmunol.org 6116 INDUCTION OF CTL IMMUNITY BY ALVAC VACCINE

Medical Center, Leiden, The Netherlands). The experiments were ap- PBS-T/1% BSA was added to the appropriate wells and incubated for 1 h proved by the animal experimental committee of Leiden University Med- at 37°C. Plates were washed four times, and 50 ␮l of ABTS (25 mg of ical Center. 3-ethyl benzthiazoline 6-sulfonic acid) dissolved in 10 ml of citrate phos- ␮ phate buffer (pH 4.2) plus H2O2 (5 l/5 ml) was added to each well. Vaccine components Absorbance values were read at 415 nm and the Ab titers were determined as the last serum dilution yielding absorbance values 5-fold higher than the Immunizations were performed with plasmid pGT64 CEA, which encodes preimmune serum. both CEA and murine CD80 (7) or ALVAC-CEA, ALVAC-CEA-CD80, or ALVAC-CEA-TRICOM. The latter two ALVAC vaccines were engi- neered to express either murine CD80 (B7-1) or a triad of murine costimu- latory molecules: CD80, ICAM-1, and CD48, the murine high-affinity li- Cytokine secretion gand of CD2. Before our comparative vaccination studies, we ensured that Splenocytes from immunized mice were incubated with 5 ␮g/ml CEA Th the ALVAC batches used did not differ essentially in quality and, in par- epitopes, CTL epitope, or inactivated ALVAC. Supernatant was analyzed ticular, that the ratio between the number of ALVAC-DNA (ge- 3 days later for Th1 and Th2 cytokines using a cytokine bead array (BD nome equivalents) and viral particles was comparable. Furthermore, by Pharmingen). infection of HeLa cells with titered amounts of different ALVAC preparations and measuring CEA expression levels, we have shown that the batches of ALVAC-CEA, ALVAC-CEA-CD80, and ALVAC-CEA-TRICOM used in our immunization experiments induced CEA expression on infected Results cells with comparable efficiency (data not shown). In some experiments ALVAC-CEA and DNA-CEA protect through distinct immune ALVAC was coinjected with CpG oligodeoxynucleotides (ODN) 1826, which is a 20-mer containing two CpG motifs (TTCATGACGTTCCTGA mechanisms CGTT). The latter was provided by Coley Pharmaceutical and used at 50 We have previously observed that protective immunity against the Downloaded from ␮ g/injection. CEA-overexpressing tumor MC38-CEA that is induced in For the construction of ALVAC-OVA and DNA-OVA vaccines, a gene construct encoding OVA aa 242–378 was cloned by PCR. This sequence C57BL/6 mice by immunization with ALVAC-CEA critically de- ϩ ϩ is devoid of the various signal sequences present in full-length OVA, but pends on CD4 T cells, but does not require CD8 T cells (6). b does encompasses the H-2K -restricted CTL epitope (OVA257–264) (8). As This observation was somewhat unexpected because MC38-CEA a result of the removal of the signal sequences, the OVA Ag is expressed is a nonlymphoid tumor cell line expressing MHC class I, but as a cytoplasmic protein that is not secreted (9).

ALVAC-SB contains a gene construct that encodes four defined CTL lacking MHC class II. To investigate whether the nature of this http://www.jimmunol.org/ epitopes and an Ab epitope that are arranged in a string-of-beads fashion anti-tumor immune response would be dictated by characteristics and are spaced by triple alanine sequences. We previously described this of the tumor cell MC38, the tumor Ag CEA, or the mode of vac- gene construct in the context of a recombinant adenovirus construct named cination, we compared the effect of ALVAC-CEA with that of a rAd1 (10). The sequence of the gene product is the following: MNAAA- SGPSNTPPEI-AAA-RAHYNIVTF-AAA-AIYKKSQHM-AAA-VNIRNC plasmid DNA-based vaccine encoding the same Ag (DNA-CEA). CYI-AAA-SEQKLISEEDLNN. The underlined sequences correspond to As shown in Fig. 1, B6 mice immunized with two subsequent i.m. the following epitopes, respectively: an H-2Db-restricted epitope from doses of either ALVAC-CEA or DNA-CEA rejected an otherwise the adenovirus type 5 (Ad5) E1A protein; an H-2Db-restricted epitope lethal dose of MC38-CEA tumor cells. Although we found these b from the human papillomavirus (HPV) type 16 E7 protein; an H-2k - two vaccines to protect against tumor outgrowth with similar ef- restricted epitope from the murine wild-type p53 protein; an H-2Db- restricted epitope from the Ad5 E1B protein; and a human c-Myc-de- ficiency, T cell depletion studies revealed that the underlying im- by guest on September 27, 2021 rived sequence recognized by mAb 9E10 (10). mune effector mechanisms differed considerably. In accordance with our previous report, in which we administered ALVAC-CEA Immunizations and tumor challenge experiments through the i.v. route (6), the antitumor efficacy of i.m. ALVAC- Mice were vaccinated twice i.m. within a 2-wk interval with 100 ␮gof CEA vaccination was abolished by in vivo depletion of the CD4ϩ 7 plasmid pGT64 CEA B7-1 (7) or with 3 ϫ 10 PFU ALVAC-CEA (pro- T cell subset, whereas depletion of the CD8ϩ T cell subset did not vided by Sanofi Pasteur) dissolved in 100 ␮l of PBS. At 4 days before each immunization, 80 ␮lof10␮M cardiotoxin was injected i.m. Two weeks diminish its protective effect (Fig. 1A). In contrast, the antitumor after the last vaccination, were isolated for in vitro tests or mice efficacy of DNA-CEA vaccination was severely compromised by ϩ ϩ were used for tumor challenge experiments in which 250,000 MC38-CEA depletion of either the CD4 or the CD8 T cell subset (Fig. 1B). tumor cells were s.c. injected in 200 ␮l of PBS/0.5% BSA. MC38-CEA In view of the differences in antitumor effector mechanisms in- cells (11) were cultured as previously described (6). CEA expression by duced by ALVAC-CEA and DNA-CEA, we dissected the CEA- MC38-CEA was regularly examined by cell surface staining with a CEA- specific Ab (PARLAM4, Monosan). In vivo depletion of CD4ϩ, CD8ϩ, specific T cell responses elicited by these vaccines. The reactivity Gr-1ϩ, or NK1.1ϩ cells involved weekly i.p. injections of 25 ␮g of GK1.5, of splenocytes from vaccinated mice was assayed against a pool of 25 ␮g of mAb 2.43, 200 ␮g of RB6-8C5, or 100 ␮g of PK136 Ab, re- five synthetic 25-mer peptides that comprise previously defined spectively, starting 1 wk before the tumor challenge and continued during I-Ab-restricted CD4ϩ Th epitopes (6), and against a synthetic the experiment. 9-mer peptide that represents a previously defined H-2Db-re- In vitro analysis of T cell responses stricted CD8ϩ CTL epitope (12) (data not shown). The CEA-spe- ϩ ϩ Splenocytes from immunized mice were tested for their responsiveness cific responses by the CD4 and CD8 T cell subsets were dis- against CEA Th epitopes 1–5 (6), the CTL epitope with peptide sequence cerned by combining intracellular staining for IFN-␥ with surface CGIQNSVSA (12) and protein by flow cytometry. A total of 2 ϫ 106 staining for CD4 and CD8. ALVAC-CEA vaccination induced splenocytes were incubated with 5 ␮g/ml Ag and brefeldin A (10 ␮g/ml) CD4ϩ T cell responses measurable in the presence of the Th for 12–16 h. Fixation and staining procedures were done as previously epitope pool, but this regimen consistently failed to induce any described (13). CD8ϩ T cell immunity against the CTL epitope (Fig. 2). In Ab detection contrast, CEA-specific responses induced by the DNA-CEA ϩ ϩ CEA-specific Abs were measured in sera from immunized mice 2 wks after vaccine involved both CD4 and CD8 T cells (Fig. 2). The in the last vaccination. Nunc Maxisorp immunoplates were coated with 2 vitro T cell data closely match those of the tumor challenge ␮g/ml CEA protein diluted in coating buffer and incubated overnight at experiments. ALVAC-CEA induces CEA-specific CD4ϩ T cell room temperature. Nonspecific binding sites were blocked with 50 ␮lof immunity only and its protective antitumor effect depends on PBS-0.05% Tween 20 (PBS-T)/1% BSA and incubated for1hat37°C. ϩ ϩ ␮ CD4 T cells, not on CD8 T cells. DNA-CEA induces CEA- After four washes with PBS-T, 50 l of serial dilutions of mouse serum in ϩ ϩ PBS-T/1% BSA was added and the plates were incubated for1hat37°C. specific CD4 and CD8 T cells, and the antitumor efficacy of After washing as described, 50 ␮l of detection Ab in diluted 1/500 in this vaccine depends on both T cell subsets. The Journal of Immunology 6117 Downloaded from http://www.jimmunol.org/ FIGURE 1. Involvement of CD4ϩ and CD8ϩ T cell subsets in antitumor effect of ALVAC-CEA and DNA-CEA. C57BL/6 mice (15–16 per group) were i.m. vaccinated twice at a 2-wk interval with 3 ϫ 107 PFU of ALVAC-CEA (A) or with 100 ␮g of DNA-CEA (B). Two weeks after the last vaccination, mice were s.c. challenged with a dose of 2.5 ϫ 105 MC38-CEA cells. Where indicated, mice were depleted for CD8ϩ or CD4ϩ T cells, depletion starting 5 days before tumor challenge. Tumor size was measured every 3 days and mice were sacrificed when tumor size exceeded 100 mm2. Each line in the graph represents tumor growth over time in a single mouse. The horizontal lines represent one or more mice in which no tumor growth was detected. The numbers at the right of these lines indicate the fraction of mice in each group that were tumor-free at the end of the experiment. CD4 depletion significantly reduced antitumor efficacy of ALVAC-CEA vaccination (p Ͻ 0.02 log-rank test), whereas both CD4 and CD8 depletion significantly affected antitumor efficacy of DNA-CEA vaccination (p Ͻ 0.003 and p Ͻ 0.009, respectively). by guest on September 27, 2021 Immune effector mechanisms mediating antitumor protection by the tumor cell surface, we tested the involvement of the CEA- ALVAC-CEA specific humoral response. We found ALVAC-CEA immuniza- The protective effect of ALVAC-CEA vaccination does not rely tion to induce high levels of anti-CEA serum IgG, primarily of on MHC class I-restricted CD8ϩ T cell immunity, arguing that the IgG1 and IgG2a subtypes (Fig. 3). Vaccination with DNA- other CEA-specific effector mechanisms must play a crucial CEA induced much lower titers of anti-CEA IgG, which were role in controlling tumor growth. Because CEA is expressed at primarily of the IgG2a subtype (Fig. 3). To analyze whether the

FIGURE 2. CEA-specific CD4ϩ and CD8ϩ T cell responses induced by vaccination with ALVAC-CEA or DNA-CEA. A, C57BL/6 mice were i.m. immunized twice at a 2-wk interval with 3 ϫ 107 PFU ALVAC-CEA or 100 ␮gof DNA-CEA. Splenocytes were isolated 2 wk af- ter the last vaccination and assayed for IFN-␥ production by intracellular cytokine staining in the presence of synthetic CEA peptide epitopes. Horizontal bar depicts the mean percentage of IFN-␥-producing CD4ϩ/CD8ϩ T cells of a group of five mice. B, Dot plots of representative examples of each group of the experiment shown in A. 6118 INDUCTION OF CTL IMMUNITY BY ALVAC VACCINE

CEA-vaccinated mice through treatment with an NK1.1-spe- cific Ab abolished the protective antitumor effect of this vaccine (Fig. 5A). In contrast, depletion of Gr-1ϩ leukocytes including neutrophils with a Gr-1-specific Ab did not significantly reduce antitumor efficacy of the ALVAC-CEA vaccine. Because Abs also play a crucial role in activating the complement system, we assessed whether mice lacking C1q, an upstream component of the classical complement activation cascade, would be able to induce an effective antitumor response upon vaccination with ALVAC-CEA. C1q knockout mice vaccinated with ALVAC- CEA and challenged with MC38-CEA showed significantly bet- ter survival than nonvaccinated C1q knockout mice (Fig. 5B), indicating that the complement cascade played no more than a minor role in tumor eradication. Taken together, our data demonstrate that the antitumor effects FIGURE 3. CEA-specific IgG responses induced by vaccination with of ALVAC-CEA and DNA-CEA vaccination rely on clearly dis- ALVAC-CEA or DNA-CEA. C57BL/6 mice were immunized i.m. twice at tinct immune mechanisms. CD8ϩ T cells are crucial for tumor ϫ 7 ␮ a 2-wk interval with 3 10 PFU ALVAC-CEA or 100 g of DNA-CEA, eradication in DNA-CEA vaccinated mice. In contrast, the antitu- respectively. Blood was drawn from the tail vein 2 wk after the last vac- ϩ mor efficacy of ALVAC-CEA vaccination depends on CD4 T Downloaded from cination, and serum IgG titers were measured by ELISA. Data depict the mean IgG titer of each group of six mice. cells, Fc receptor function and NK cells, suggesting that ADCC is the main effector mechanism involved. high levels of CEA-specific IgG induced by ALVAC-CEA con- Impact of the ALVAC vector on CEA-specific immunity tributed to the antitumor effector response, we performed tumor The distinct antitumor effector mechanisms induced by ALVAC- ␥ challenge experiments in Fc receptor -chain knockout mice. CEA and DNA-CEA vaccination prompted us to examine the http://www.jimmunol.org/ Due to the absence of the common ␥-chain, these mice lack cytokine profile of the CEA-specific T cell responses. Because functional expression of the activating Fc receptors Fc␥RI, ALVAC-CEA comprises multiple canarypox virus proteins, we Fc␥RIII, Fc␥RIV (and Fc␧RI) that are involved in, among other also analyzed the vaccine-induced response against these vector activities, Ab-dependent cellular cytotoxicity (ADCC) and Fc components. Splenocytes from mice immunized with either ALVAC- receptor-mediated release of inflammatory mediators (14). Fig. CEA or DNA-CEA were cultured in the presence of CEA peptide 4 shows that the antitumor efficacy of ALVAC-CEA vaccina- epitopes or inactivated ALVAC, after which the culture superna- tion is greatly reduced in Fc receptor ␥-chain knockout mice, tants were analyzed for the presence of secreted cytokines. Cyto- whereas the protective effect of the DNA-CEA vaccine is not kine bead array analysis of Th1/Th2 cytokines showed that in par- significantly affected. These results suggest that tumor eradica- ticular the Th1 cytokine IFN-␥ and the Th2 cytokine IL-5 were by guest on September 27, 2021 tion in ALVAC-CEA vaccinated mice is dependent on ADCC. secreted. In accordance with the data in Fig. 2, DNA-CEA vacci- We therefore examined the role in the antitumor response of NK nation induced CEA-specific T cell activity against both the CD4ϩ cells and neutrophils, both of which express Fc␥RIII and can Th epitopes and the CD8ϩ CTL epitope, whereas ALVAC-CEA become activated upon binding of immune complexes compris- vaccination induced immunity against the CD4ϩ Th epitopes only. ing IgG1 and IgG2a (14). Indeed, NK depletion of ALVAC- Furthermore, as anticipated, ALVAC-CEA induced very strong T

FIGURE 4. Antitumor efficacy of ALVAC-CEA vaccination is depen- dent on Fc receptor ␥-chains. C57BL/6 wild-type (A) and Fc receptor ␥-chain knockout (B) mice were immunized and challenged with MC38-CEA tumor cells as described in Fig. 1. Lines rep- resent tumor growth over time in one mouse. Each horizontal line represents one or more mice in which no tumor growth was detected. The number at the right of this line indicates the frac- tion of mice in each group that were tumor-free at the end of the experiment. DNA-CEA vaccination resulted in sig- nificant protection of Fc receptor ␥-chain knockout mice from tumor growth as compared with ALVAC- CEA-vaccinated and nonvaccinated groups (p Ͻ 0.003 and 0.0001, respec- tively). This experiment was performed twice with comparable outcomes. The Journal of Immunology 6119 Downloaded from http://www.jimmunol.org/

FIGURE 5. Important role for NK1.1ϩ cells, but not for neutrophils and complement, in antitumor efficacy of ALVAC-CEA vaccination. A, C57BL/6 wild-type (A) and C1q knockout (B) mice were immunized with ALVAC-CEA and challenged with MC38-CEA tumor cells as described in Fig. 1. Each line in the graph represents tumor growth over time in one mouse. The horizontal line represents one or more mice in which no tumor growth was detected. The number at the right of this line indicates the fraction of mice in each group that were tumor-free at the end of the experiment. Where indicated, mice were depleted for Gr-1ϩ cells or NK1.1ϩ cells, depletion starting 5 days before tumor challenge. ALVAC-CEA vaccination resulted in significant reduction of tumor development in C1q knockout mice (p Ͻ 0.003), C57BL/6 wild-type mice (p Ͻ 0.003), and C57BL/6 wild-type mice that underwent Gr-1

depletion (p Ͻ 0.003). by guest on September 27, 2021

cell reactivity against ALVAC vector components (Fig. 6). Inter- estingly, the responses against CEA Th epitopes induced by DNA- CEA were associated with IFN-␥ secretion only, whereas the re- sponses against CEA Th epitopes induced by ALVAC-CEA involved secretion of both IFN-␥ and IL-5. This mixed Th1/Th2 cytokine profile was also found for the ALVAC-specific responses induced by ALVAC-CEA (Fig. 6). The cytokine profiles of the responses elicited by DNA-CEA and ALVAC-CEA correspond very well with the immune effector responses that mediate the antitumor effect of these vaccines. The efficacy of DNA-CEA vac- cination involves the induction and action of CEA-specific CD8ϩ T cells (Fig. 1), and accordingly this immune response is associ- ated with a Th1-type cytokine profile that optimally supports the induction and maintenance of CTL immunity. In contrast, the ef- ficacy of ALVAC-CEA vaccination involves the induction and action of CEA-specific CD4ϩ T cells and IgG Abs (Figs. 3–5), and accordingly this immune response features a mixed Th1/Th2 pro- file that is more supportive of humoral responses. The virus-specific responses induced by ALVAC-CEA are markedly stronger than the CEA-specific responses (Fig. 6). It is therefore conceivable that the character and polarization of the immune response against CEA is influenced by the dominant re- FIGURE 6. Specificity and cytokine profile of T cell response induced by sponses against ALVAC Ags. We initially considered the possi- ALVAC-CEA or DNA-CEA. C57BL/6 mice were immunized with ALVAC- bility that immunization with lower amounts of ALVAC might CEA or DNA-CEA as described in Fig. 1. Splenocytes were isolated 2 wks result in a lower viral load and, thereby, might favor the attention after the last vaccination and incubated for 3 days with synthetic CEA peptide of the immune system toward the transgene-encoded Ag CEA. epitopes or inactivated ALVAC. Culture supernatants were tested for the pres- However, immunizations with lower ALVAC-CEA titers did not ence of cytokines by cytokine bead array analysis. Data are the mean cytokine ϩ ϩ result in the enhancement of CEA-specific CD4 and/or CD8 T secretion of each group of three mice, shown for IFN-␥ and IL-5. 6120 INDUCTION OF CTL IMMUNITY BY ALVAC VACCINE

FIGURE 7. The cytokine profile induced by ALVAC-CEA cannot be changed by several modi- fications of the vaccine. C57BL/6 mice were immu- nized with ALVAC-CEA, DNA-CEA, or a combi- nation of ALVAC empty vector and DNA-CEA (A), as described in Fig. 1. Mice immunized with both vac- cines received the combination in both limbs (DNAϩALVAC) or separate injections of ALVAC and DNA-CEA in the left and right limb, respectively (ALVAC/DNA). Similar immunizations were also performed with ALVAC-CEA-CD80 and ALVAC- CEA-TRICOM (B). Analysis of Ag-specific T cell re- activity was assayed for IFN-␥ production by intracel- lular cytokine staining in the presence of synthetic CEA peptide epitopes as described for Fig. 2A. Data shown are the mean percentage of each group of three mice. Results are from two separate experiments with similar outcome. Downloaded from

cell responses (data not shown). To further investigate the impact the genes for all three aforementioned murine costimulatory pro- of ALVAC-CEA on polarization of T cell immunity, mice were teins (ALVAC-CEA-TRICOM) (see Materials and Methods). As coimmunized with DNA-CEA and ALVAC control vector, and the shown in Fig. 7B, we found the CEA-specific T cell responses

cytokine profiles of the CEA and ALVAC-specific responses were induced by vectors encoding either CD80 or TRICOM to be in- http://www.jimmunol.org/ analyzed. Coinjection of ALVAC largely precluded the induction distinguishable from those elicited by the parental ALVAC-CEA of CEA-specific CTL responses by DNA-CEA, and also resulted vaccine. in lower CEA-specific Th1 responses (Fig. 7A). Similar results Our data demonstrate that the ALVAC vector has a profound were obtained when coimmunization experiments were performed impact on the character of the immune response against the trans- with ALVAC-CEA instead of the ALVAC empty vector (data not gene-encoded CEA, which cannot be overruled by either coinjec- shown). Notably, this effect of ALVAC coinjection was not only tion of DNA vaccine or CpG oligodeoxynucleotides, nor by com- observed when ALVAC and DNA-CEA were administered as a plementing ALVAC vaccines with genes encoding powerful mixture in the same site (Fig. 7A, ALVACϩDNA), but also when costimulatory molecules. these vaccines were injected separately in opposite limbs (Fig. 7A, by guest on September 27, 2021 ALVAC/DNA). Thus, the impact of ALVAC on the character and ALVAC immunization elicits CTL immunity against highly polarization of the CEA-specific response is not confined to the immunogenic epitopes local draining lymph nodes, but also acts at the systemic level. Although none of the ALVAC-CEA immunization protocols Furthermore, this effect does not require linkage of the CEA and tested elicited CEA-specific CD8ϩ T cell immunity, the ALVAC- ALVAC Ags within a single vaccine formulation or vector. induced Th1/Th2 profile does not appear to be prohibitive for CTL One of the reasons why DNA-CEA vaccine is more efficient in activation, in that the T cell responses against the ALVAC vector inducing Th1/CTL type immunity than ALVAC-CEA may be that component, although dominated by CD4ϩ T cell immunity, com- naked plasmid DNA is rich in CpG sequences that can trigger prised an, albeit modest, CD8ϩ component (Fig. 8A). This finding innate immunity through TLR9. Especially in C57BL/6 mice, suggested that the capacity of ALVAC vaccines to induce CTL TLR9 ligands, such as synthetic oligomers comprising CpG oli- immunity may be limited to highly immunogenic CTL epitopes, godeoxynucleotides sequences, were shown to constitute excellent such as those comprised by the . We therefore studied vaccine adjuvants for the induction of Th1/CTL immunity (see the impact of the ALVAC context on the immune response against references in Ref. 15). However, our finding that a mixture of the commonly used model Ag OVA, which contains a potent b ALVAC-CEA with DNA-CEA vaccine did not result in enhance- H-2K -restricted CTL epitope (OVA257–264) (8). Interestingly, we ment of the CEA-specific Th1/CTL response (Fig. 7A), suggested found ALVAC-OVA to elicit a very strong T cell response against that in vivo stimulation of TLR9 at the vaccination site could not the OVA CTL epitope (Fig. 8B). The ALVAC-OVA-immunized overrule the dominant features of ALVAC-induced immunity. Ac- mice, like their ALVAC-CEA-immunized counterparts, did show cordingly, we found that a mixture of ALVAC-CEA with CpG a strong T cell response against ALVAC that was associated with oligodeoxynucleotides similarly did not result in increased CEA- the secretion of both IFN-␥ and IL-5 (data not shown). In case of the specific Th1/CTL immunity (data not shown). Reports by others OVA Ag, however, the presence of this ALVAC-specific Th1/Th2 have suggested that incorporation into the vector of genes that response did not preclude the induction of a very strong CTL re- encode costimulatory molecules, such as CD80 (B7-1), ICAM-1, sponse. On the contrary, the immune context provided by ALVAC and CD2 ligand (human LFA-3 or murine CD48), can improve the vaccination appears to be optimal for induction of OVA-specific CTL antitumor efficacy of ALVAC and other poxvirus-based CEA vac- immunity, in that these responses exceed those induced by an OVA- cines (see references in Ref. 16). Furthermore, the CEA-encoding encoding DNA vaccine or synthetic peptide-based formulations that plasmid DNA used in our experiments (DNA-CEA) also encom- we tested in parallel (data not shown). passes the gene-encoding murine CD80 (see Materials and Meth- Further evidence of the capacity of ALVAC vaccines to elicit ods). We therefore examined the characteristics of CEA-specific T Ag-specific CTL immunity was obtained through immunizations cell responses induced by ALVAC-CEA vaccines that incorpo- with a construct comprising a string-of-beads arrangement of four rated either the gene for murine CD80 (ALVAC-CEA-CD80) or well-defined CTL epitopes (ALVAC-SB), derived from the human The Journal of Immunology 6121

the predisposition of ALVAC-based vaccines to elicit a mixed Th1/Th2 profile should not be interpreted as an immune escape mechanism, but rather as an intrinsic feature of the natural immune response against this virus. Importantly, these Th1/Th2 responses are largely directed against ALVAC vector Ags (Fig. 6). It is con- ceivable that the character of the CEA-specific immune response is dictated by that of the dominating response against the vector Ags, particularly when the transgene-encoded Ag does not comprise highly immunogenic CTL epitopes. The CEA CTL epitope CGIQNSVSA (12), although containing the anchor residues com- patible with H-2Db binding, displays only intermediate MHC class I binding (data not shown). Furthermore, CEA is a glycoprotein, and this could further limit efficient presentation of its CTL FIGURE 8. Induction of CTL immunity by ALVAC-based vaccines. epitope. For another glycosylated Ag, MUC-1, the efficiency of C57BL/6 mice were immunized with ALVAC-CEA (A), ALVAC-OVA processing into MHC class I and the resulting strength of CTL (B), or ALVAC-SB (C) as described in the Fig. 1. Splenocytes were iso- immunity were shown to be inversely correlated with the degree of lated 2 wks after the last vaccination, after which the cells were directly assayed for IFN-␥ production by overnight intracellular cytokine staining glycosylation (20). Notably, the availability of highly immuno- ϩ ϩ genic T cell epitopes within a given Ag depends on the repertoire in the presence of CEA CD4 and CD8 peptide epitopes, inactivated Downloaded from ALVAC (A)orCD8ϩ peptide epitopes derived from OVA, Ad5 E1A, of MHC molecules expressed by the immunized subject, implying HPV16 E7, wild-type murine p53, or Ad5 E1B (B and C). The latter four that vaccination with ALVAC-CEA may elicit CEA-specific CTL epitopes are encoded by ALVAC-SB, as described in Materials and Meth- responses in mice strains expressing a different set of MHC class ods. Data are the mean results of each group of 3 (A)and5(B and C) mice, I molecules. respectively. Our present studies were performed in mice for which CEA

is a foreign Ag, suggesting that induction of CEA-specific T http://www.jimmunol.org/ Ad5 Ags E1A and E1B, from the HPV16 E7 Ag, and from wild- cell immunity by the vaccines studied would be more difficult in type murine p53 (see Materials and Methods). As shown in Fig. a host that expresses CEA as an autoantigen. Indeed, we found 8C, ALVAC-SB elicits CTL responses against the Ad5 E1A and that ALVAC-CEA vaccination of CEA transgenic mice only elic- E1B epitopes, but not against the HPV16 E7 and p53 epitopes. ited weak CD4ϩ T cell immunity against subdominant CEA CD4ϩ This result is in concordance with our previous findings that the T cell epitopes distinct from those detected in nontransgenic mice E1A and E1B epitopes were highly immunogenic, whereas the E7 (6). This difference in CEA-specific T cell responses is largely due epitope was much less immunogenic, and a proper repertoire for to clonal deletion of CEA-specific T cells in the of the the p53 epitope is lacking in normal mice as a result of self-tol- CEA transgenic mice (6). The magnitude and breadth of these T erance (17–19). cell responses in CEA transgenic mice was not significantly in- by guest on September 27, 2021 In conclusion, the capacity of ALVAC-based vaccines to elicit creased by applying various homologous or heterologous prime- ϩ CD8 T cell immunity against the transgene-encoded Ag of in- boost protocols that involved CEA-specific vaccines based on pro- terest strongly depends on the nature of this Ag, in particular on the tein, DNA, ALVAC, or virus. Accordingly, these presence of highly immunogenic CTL epitopes in the Ag. vaccination regimens were ineffective in causing rejection of MC38-CEA tumors in CEA transgenic mice (our unpublished Discussion data). Paradoxically, work by other researchers showed that opti- ALVAC and DNA-based vaccines encoding the tumor Ag CEA mized immune intervention protocols involving vaccination with elicit Ag-specific immune responses with distinct characters. CEA-encoding poxviruses did protect these CEA transgenic mice Whereas DNA-CEA induces Th1/CTL-type immunity, ALVAC- against outgrowth of CEA-positive tumors (16, 21, 22). However, CEA induces Th1/Th2-responses that are associated with strong these studies did not involve detailed analysis of the CEA-specific humoral immunity. The propensity of ALVAC-CEA to elicit Th1/ T cell responses. It is therefore unclear to which extent CEA- Th2-type immunity, instead of a Th1/CTL response, could not be specific immunity contributed to the antitumor effects observed. In changed by modifying the vaccine through coinjection of DNA-CEA, fact, recent papers indicated that T cell responses against a foreign, admixture with CpG oligodeoxynucleotides, or supplementation with retroviral epitope contributed significantly to this antitumor im- additional transgenes encoding costimulatory molecules. Even though mune response (21–23). In contrast, work by a yet another labo- our experiments with ALVAC-CEA suggested that ALVAC-based ratory demonstrated that CEA-specific vaccination of CEA trans- vaccines may not be suitable for inducing CTL immunity against genic mice, using DNA and recombinant adenovirus-based transgene-encoded Ags, immunization with ALVAC-OVA resulted ϩ ϩ in impressive OVA-specific CTL responses. Furthermore, immuniza- vaccines, did elicit clear-cut CEA-specific CD8 and CD4 T cell tion with ALVAC-SB, comprising a minigene, which encodes a immunity that was instrumental in preventing outgrowth of CEA string-of-beads arrangement of CTL epitopes, resulted in induction of overexpressing tumors (12, 24, 25). Notably, the CEA transgenic CTL immunity against the two most immunogenic peptides within mice used by Primus and colleagues (11) express lower levels of this multi-epitope construct. Therefore, the Th1/Th2 cytokine profile CEA than mice used by our group (26), suggesting that the CEA- induced by ALVAC immunization is not prohibitive for induction of specific T cell repertoire in the former mice was less affected by CTL immunity, provided the CTL epitopes concerned are highly self-tolerance. immunogenic. In conclusion, our data demonstrate that the capacity of ALVAC The finding that ALVAC-based vaccine triggers mixed Th1/Th2 vaccines to elicit CTL immunity against transgene-encoded Ags immunity, as opposed to responses with a strong Th1 bias, can depends on the presence of immunogenic CTL epitopes in such most likely be attributed to the fact that both cellular and humoral Ags. In the case of Ags with strong CD8ϩ T cell epitopes, ALVAC immunity are important for combating viral infections. Therefore, can induce high levels of CTLs, but in the case of Ags with weaker 6122 INDUCTION OF CTL IMMUNITY BY ALVAC VACCINE epitopes, the Th1/Th2 bias of ALVAC may limit the CTL re- 11. Clarke, P., J. Mann, J. F. Simpson, K. Rickard-Dickson, and F. J. Primus. 1998. sponse. The latter limitations cannot be overcome by supplemen- Mice transgenic for human carcinoembryonic antigen as a model for immuno- therapy. Cancer Res. 58: 1469–1477. tation of ALVAC with genes encoding costimulatory molecules, or 12. Mennuni, C., F. Calvaruso, A. Facciabene, L. Aurisicchio, M. Storto, E. Scarselli, admixture with strong Th1-polarizing adjuvants such as CpG G. Ciliberto, and N. La Monica. 2005. Efficient induction of T-cell responses to carcinoembryonic antigen by a heterologous prime-boost regimen using DNA ODN. This information is highly relevant for the various ongoing and adenovirus vectors carrying a codon usage optimized cDNA. Int. J. Cancer vaccine development programs against cancer and infectious dis- 117: 444–455. eases that make use of ALVAC or closely related viral vectors as 13. van der Burg, S. H., K. M. Kwappenberg, T. O’Neill, R. M. Brandt, C. J. Melief, J. K. Hickling, and R. Offringa. 2001. Pre-clinical safety and efficacy of TA-CIN, delivery tools (1, 2, 4). a recombinant HPV16 L2E6E7 fusion protein vaccine, in homologous and het- erologous prime-boost regimens. 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