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Immunology |
Laboratory of Tumor Immunology and Biology, National Cancer Institute [D. W. G., J. S., J. W. H.] and Howard Hughes Medical Institute, Research Scholars at the NIH [J. C. B.], NIH, Bethesda, Maryland 20892-1750
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most experimental and some clinical vaccine studies to date have used various strategies to enhance T-cell responses to specific TAAs. Among these are: (a) the use of vectors for more efficient delivery of the TAA to the APC (9, 10, 11, 12) and for more efficient processing with MHC molecules (13 , 14) ; (b) the use of T-cell costimulation either by antibody-mediated blockade mechanisms (15 , 16) or via inserting costimulatory molecules into vectors (17, 18, 19) ; (c) the use of cytokines to enhance either APC function or T-cell function (20, 21, 22, 23, 24, 25) ; and (d) the use of diversified prime and boost vaccine strategies (26, 27, 28, 29, 30, 31, 32) . Although several studies have used two or more of the above vaccine strategies, few studies, if any, have analyzed multiple strategies to determine maximum ability to activate T cells.
In previous experimental studies, we and others have demonstrated the following: (a) The use of recombinant orthopox vectors such as vaccinia or avipox [fowlpox and/or canarypox (ALVAC)] to enhance T-cell responses to TAAs and to induce antitumor immunity as compared to the use of the TAA protein in adjuvant (5 , 27) . (b) Diversified vaccine prime and boost regimens were advantageous in enhancing T-cell responses. A rV vector cannot be used multiple times because of the induction of host immunity to the virus (33, 34, 35, 36) . However, it has been shown that a primary vaccination with a rV vector followed by booster vaccinations with recombinant avipox viruses leads to optimal induction of T-cell responses (32) . In these studies and others, it has been shown that the use of two different recombinant vectors, in prime and boost strategies, can be more potent in inducing T-cell responses than the continued use of one single vector. (c) The insertion of genes for a costimulatory molecule in vectors also containing the gene for a TAA enhances T-cell responses to the TAA. Moreover, the use of rV or rF vectors containing a triad of costimulatory molecules (B7-1, ICAM-1, LFA-3; designated TRICOM) has been shown to activate T cells to greater levels than the use of any one or two of these costimulatory molecules in recombinant vectors (18) . (d) The use of cytokines such as GM-CSF has been shown to enhance the infiltration of APC, including dendritic cells, to regional nodes and consequently enhance T-cell responses when given for 4 consecutive days following vaccine (5 , 37) . More recently, it has been shown that a single administration of rF-GM-CSF given with vaccine is as potent as four daily administrations of recombinant GM-CSF in enhancing T-cell responses (38) . In addition, it has been previously shown that when IL-2 has been used in low doses several days following vaccination, antigen-specific T-cell responses can be further enhanced (25) .
Although the vast majority of experimental vaccine studies in mouse models have used conventional mice, and the target antigen is not "self," most human TAAs identified to date have been shown to be "self-antigens," with some expression on normal adult tissues. CEA is a TAA, which is overexpressed in most carcinomas, including gastrointestinal carcinomas, and is also expressed at lower levels in normal colonic mucosa (39) . The most common site of metastases of CEA-positive malignancies in patients is the liver; CEA protein is also shed into the serum of many patients with metastatic CEA-positive malignancies (39) . There are several clinical studies that have now demonstrated that one can elicit CEA-specific T-cell responses in patients vaccinated with CEA-based vaccines (4 , 40, 41, 42, 43, 44, 45, 46) . Studies have also demonstrated that the CEA-specific T cells generated are capable of lysing CEA-expressing carcinoma cells in a MHC-restricted manner (4 , 41, 42, 43 , 47, 48, 49) . Although indications of prolonged survival and some antitumor responses have been reported (42 , 43) , the main issue may well be the potency of the T-cell responses that have been generated in metastatic cancer patients to achieve therapeutic responses.
Many of the experimental studies described to date have used conventional mice to demonstrate antitumor immunity. A more appropriate model would be the use of mice in which the TAA is a self-antigen, such as CEA, with expression on some normal adult tissues. CEA-transgenic mice express CEA in normal adult gastrointestinal tissues in a manner similar to that expressed in humans and also express relatively high levels of CEA protein in serum, i.e., at levels seen in many patients with advanced colon carcinoma (50) . Moreover, CEA-transgenic mice have previously been shown to be tolerant to the induction of CEA-specific T-cell or antibody responses following vaccination with CEA protein in adjuvant (5) . One study has shown that the administration of rV-CEA can reduce the growth rate of CEA-expressing tumors transplanted s.c. (25) . Like many animal models published to date (17 , 20 , 51, 52, 53, 54, 55) , the antitumor effect of the vaccine was seen only when vaccine therapy was initiated within 7 days posttumor transplant; waiting longer periods of time to initiate therapy was not successful.
In the studies described here, we have used these multiple vaccine strategies in an effort to obtain high levels of T-cell responses to a target antigen, CEA. Because of the limited availability of CEA-transgenic mice to conduct all of the studies reported here, initial studies were carried out in conventional mice to define optimal parameters of vaccine strategies. CEA-transgenic mice were then used to verify the induction of immune responses specific for CEA and in tumor therapy studies in which the TAA is a self-antigen. Using the multiple vaccine strategies described above, antitumor studies were carried out in the studies described here in CEA-transgenic mice bearing 14-day experimental CEA-expressing liver metastases.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals/Cells.
Female C57BL/6 mice were obtained from the National Cancer Institute, Frederick Cancer Research Facility (Frederick, MD). Mice were housed and maintained under pathogen-free conditions in microisolator cages until used for experiments at 6–8 weeks of age. Maintenance of experimental mice continued under the same conditions. C57BL/6 mice transgenic for human CEA mice were obtained from a breeding pair provided by Dr. John Thompson (Institute of Immunobiology, University of Freirburg, Freirburg, Germany). The generation and characterization of the CEA-transgenic mouse has been previously described (50)
. Murine colon adenocarcinoma cells expressing human CEA (MC-38-CEA-2) have been described elsewhere (60)
. Prior to transplantation to mice, the cells were trypsinized and washed twice in HBSS before final suspension in the same.
Vaccine Dose and Route Studies.
Six- to 8-week-old female C57BL/6 mice were vaccinated i.v. or s.c. with (a) 100 µl of HBSS; (b) either 108 or 109 pfu rF-CEA; or (c) either 108 or 109 pfu rF-CEA/TRICOM. All viruses used here and in subsequent experiments were diluted in HBSS such that the entire dose was delivered in 100 µl. Fourteen and 28 days later, mice were boosted using identical reagents. Fourteen days following the final vaccination, CEA-specific T-cell responses were analyzed as previously described (61)
.
Titration of Serum Antibodies.
Anti-CEA and anti-fowlpox antibody (IgG) were quantified in the serum of each animal by ELISA. Microtiter plates were coated with either purified CEA (100 ng/well), FP-WT (1 x 107 pfu/well), or ovalbumin (100 ng/well). The plates were blocked with 5% BSA in PBS, dried, and stored at -20°C until used. The plates were incubated with serum serially diluted from 1:50 to 1:6250, as well as COL-1 or rabbit antifowlpox antiserum as controls, for 24 h at 4°C. Plates were washed several times with PBS containing 1% BSA and incubated at 37°C for 45 min with horseradish peroxidase-conjugated goat antimouse IgG-specific antiserum (1:4000) and antibody was detected by a horseradish peroxidase substrate system (Kirkegaard & Perry Laboratories, Gaithersburg, MD) according to the manufacturer’s instructions. The absorbance of each well was read at 490 nm using a Bio-Tek EL310 microplate ELISA reader (Winooski, VT). Antibody titers were based on an absorbance of 1.5.
Prime and Boost Studies.
C57BL/6 mice were vaccinated s.c. with 108 pfu of either rV-CEA/TRICOM or rF-CEA/TRICOM. Fourteen days later, mice were boosted with 108 pfu of rF-CEA/TRICOM. In a parallel experiment, mice were vaccinated with 108 pfu V-WT 14 days prior to beginning the above described vaccination strategies. Fourteen days following the final vaccination, CEA-specific T-cell responses were analyzed as described above.
rF-GM-CSF Studies.
C57BL/6 mice were vaccinated s.c. with 108 pfu of either rF-CEA or rF-CEA/TRICOM alone or admixed with 107 pfu of rF-GM-CSF. Fourteen days later, CEA-specific T-cell responses were analyzed as described above.
Tumor Therapy Studies.
CEA-transgenic mice were transplanted with MC38-CEA-2 cells as experimental hepatic metastases. The procedure for transplantation of hepatic metastases has been described (62)
. This dose of tumor cells (50,000) is lethal to >80% of mice within 12 weeks. Fourteen days following tumor transplant, mice were vaccinated as described in the legends to Figs. 6
and 7
. The therapy studies were conducted twice with similar results, and the data shown in Fig. 7
are a composite of the results of both experiments.
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by capture ELISA as described previously (65)
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Statistical Analysis of the Data.
Where indicated, the results of tests of significance are reported as P and are derived from Student’s t test using a two-tailed distribution. Evaluation of survival patterns in mice bearing hepatic tumors was performed by the Kaplan-Meier method and ranked according to the Mantel-Cox log rank test using a Statview 4.1 (Abacus Concepts, Berkeley, CA) software package. For graphical representation of data, y-axis error bars indicate the SD of the data for each point on the graph. The y-axis error bars in Fig. 3
are reported as SE because the data are averaged from three individual mice analyzed in triplicate.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the administration of rF-CEA/TRICOM was clearly more effective in inducing CEA-specific proliferative T-cell responses when given s.c. than i.v. (P = 0.026). No immune responses were seen using control vector FP-WT, and no immune responses were observed with any of the vectors to ovalbumin control protein; these studies were conducted at 108 pfu vector per vaccination. Studies were also conducted using 109 pfu vector per vaccination (Fig. 1B)
. Here again a distinct and significant difference was seen between s.c. and i.v. vaccination, with s.c. vaccination being far superior (P = 0.002). As an additional control, splenocytes from mice vaccinated with rF-CEA/TRICOM or FP-WT were also analyzed for responses to ConA. As seen in Fig. 1C
, there was a reduction in responses to ConA in mice receiving fowlpox vectors via the i.v. route. Although the reason for this effect of i.v. administered fowlpox is not known at this time, it was clear that subsequent studies should be carried out via the s.c. vaccination route.
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, three vaccinations with rF-CEA/B7-1 were clearly more potent (P = 0.01) than four vaccinations with rF-CEA in the induction of CEA-specific T-cell responses. Moreover, three vaccinations with rF-CEA/TRICOM were also more potent than three vaccinations with rF-CEA/B7-1 (P = 0.03) or four vaccinations with rF-CEA (P = 0.004) in the induction of CEA-specific T-cell responses. These studies also demonstrated that with consecutive vaccinations of rF-CEA/TRICOM CEA-specific T-cell responses continued to increase. There was no evidence of limited use of the fowlpox recombinant vector because of host antivector immune responses. This will be discussed in more detail in "Discussion." Anti-CEA IgG responses were also measured in these experiments. As seen in Fig. 3
, anti-CEA IgG responses also increased with subsequent vaccinations of either rF-CEA/B7-1 or rF-CEA/TRICOM. These studies further demonstrated the potential for multiple vaccinations of rF vectors. Note that the use of costimulatory molecules (B7-1 or TRICOM) did not significantly increase anti-CEA IgG responses as they did CEA-specific T-cell responses.
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, using rV-CEA/TRICOM as a prime vaccination followed by a boost with rF-CEA/TRICOM was significantly more potent in inducing CEA-specific T-cell responses than priming and boosting with rF-CEA/TRICOM (P = 0.048). This study was also carried out in mice that had been vaccinated 2 weeks earlier with wild-type vaccinia virus and shown to contain high levels of antivaccinia antibody (32)
. This scenario mimicked that commonly observed in the clinical situation in which most adults exhibit preexisting antivaccinia immunity as a result of prior smallpox vaccination. As seen in Fig. 4B
, the diversified prime and boost strategy of priming with rV-CEA/TRICOM and boosting with rF-CEA/TRICOM remained significantly more potent in inducing T-cell responses than the priming and boosting with rF-CEA/TRICOM (P = 0.0415). Thus, even when using vectors containing multiple costimulatory molecules, diversified prime and boost strategies appeared to be advantageous.
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, rF-GM-CSF had no effect on the induction of CEA-specific T-cell responses when administered with FP-WT. rF-GM-CSF showed a marked and significant enhancement of CEA-specific T-cell responses when used with rF-CEA (P = 0.015). This level of CEA-specific T-cell responses was similar to that achieved using the rF-CEA/TRICOM vector alone. When rF-CEA/TRICOM was used in combination with rF-GM-CSF, however, a marked difference was seen as compared to the use of rF-CEA/TRICOM alone in the induction of CEA-specific T-cell responses. As seen in Fig. 5
(inset, A), no differences in responses to ConA for any of the groups with or without rF-GM-CSF were observed. Moreover, no responses were seen in any of the groups to the negative control antigen ovalbumin.
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production was measured. A control peptide (VSV-N) was also used. As seen in Fig. 6A
, CD8+ CEA-specific responses could be observed in the CEA-transgenic mice receiving the recombinant CEA/TRICOM vaccines (group 1) which were significantly greater than those observed for the vector treatment group (group 2; P = 0.001) or the untreated group (group 3; P = 0.001). CD4+ CEA-specific responses were also observed in the group receiving the CEA/TRICOM vaccines and were significantly greater than those in the group receiving control wild-type vector vaccines with cytokines (P = 0.0013; Fig. 6B
). No significant responses were observed to the control ovalbumin protein. Concomitant to this study, a tumor therapy study was initiated to determine the therapeutic efficacy of a CEA-based vaccine in CEA-transgenic mice. In these studies, MC-38 murine colon carcinoma cells, which had been transduced with the CEA gene using a retroviral vector, were inoculated intrasplenically into CEA-transgenic mice. After dissemination of the cells into the liver through the portal vein, mice were then splenectomized; previous studies have shown that large hepatic metastases develop in similar models at 4–5 weeks posttumor transplant (66 , 67) . Mice also developed large experimental liver metastases in the model used here that were lethal in >80% of mice by 12 weeks posttransplant if left untreated. Three groups were used in these tumor therapy studies. Mice in all groups received tumor at day 0. Fourteen days after tumor transplant, vaccination was initiated in groups 1 and 2. Group 1 received a primary vaccination of rV-CEA/TRICOM followed by three weekly booster vaccinations with rF-CEA/TRICOM. This group also received recombinant GM-CSF protein with the rV-CEA/TRICOM vaccination and rF-GM-CSF with rF-CEA/TRICOM; group 1 also received low-dose systemic IL-2. Group 2 received treatment identical to group 1, except that the mice received V-WT as the primary vaccination and FP-WT for the subsequent three booster vaccinations. Group 2 also received GM-CSF and IL-2 in a manner identical to group 1. Group 3 received no treatment following tumor transplant.
As can be seen in Fig. 7
, 56.3% (9 of 16) of the mice in group 1 receiving the recombinant CEA vaccines and cytokines remained alive and apparently healthy through the 25-week observation period. However, only 5.3% (1 of 19) of the mice that received control vectors along with cytokine treatment (group 2) survived past 16 weeks. Only 8.3% (1 of 12) of the mice in the group receiving no treatment (group 3) survived past 16 weeks. The P for survival between mice receiving no treatment and treatment with control vectors plus cytokines was not significant (P = 0.43). The P for the survival of mice receiving CEA-TRICOM vaccines plus cytokines (group 1) versus the untreated group was statistically significant (P = 0.015). Furthermore, survival of mice receiving the CEA-TRICOM vectors plus cytokines was statistically significant as compared with survival of mice receiving the control vectors plus cytokines (P = 0.026).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The animal model used here for antitumor therapy employed a murine colon carcinoma cell expressing CEA to induce experimental liver metastases in which 14 days posttumor transplant vaccine therapy was initiated. The vast majority of tumor therapy models to date have initiated vaccine therapy 3–5 days posttumor transplant (14
, 17
, 31
, 51
, 68)
. To our knowledge, this is the longest time interval between tumor transplant and vaccine therapy for a tumor therapy model in which the vaccine is directed against a self-antigen. The fact that only half of the animals remained tumor free during the 23-week observation period, however, affirms the need for further studies to improve antitumor responses. These studies will involve the mechanisms of antitumor immunity as well as potential tumor escape mechanisms. More specifically, these studies will deal with histochemical and immunohistochemical analyses of the tumor site to identify both the type and quantity of effector cells and cell subsets present within the tumor infiltrate. Adoptive transfer of effector cell subsets and/or antibody-blocking experiments will best define the mechanisms of antitumor responses. The study of tumor escape mechanisms will include analysis of tumors that have escaped vaccine therapy for possible antigen loss variants, possible down-regulation of MHC molecules on tumor cells, and Fas-FasL interactions. Mice bearing tumors that have escaped vaccine therapy will also be analyzed for the presence of T cells displaying down-regulation of the 
chain of the T-cell receptor. The studies reported here do demonstrate, however, that both CD4+ and CD8+ responses specific for CEA were generated in vaccinated CEA-transgenic mice.
One of the issues that also needs to be resolved in future studies is that of pathological autoimmunity as a result of the use of vectors containing a self-antigen and costimulatory molecules. In a previous study, it was shown that repeated vaccinations with rV-B7-1 did not induce any evidence of autoimmunity (69)
. In recent toxicology studies, no evidence of autoimmunity, as analyzed by histochemistry, altered blood chemistry, or antinuclear antibody, was observed when CEA-transgenic mice received four vaccinations of rV-CEA/TRICOM or rF-CEA/TRICOM vectors. It is unclear at this time, however, if the more potent vaccine strategies used in the studies reported here in tumor-bearing animals would lead to any evidence of pathological autoimmunity. Long-term studies using CEA-transgenic mice will be required to answer this question; at the 23-week observation period in the studies reported here (Fig. 7)
, however, such mice exhibited normal body weight gain and appeared otherwise healthy.
The studies reported here demonstrated that rF-CEA and rF-CEA/TRICOM induced more potent T-cell responses when given s.c. than when administered i.v. It should also be pointed out that the i.v. dosing also reduced the ability of splenic T cells to respond to ConA mitogen. The reason for this is unclear at this time. This was seen not only with the rF-CEA and rF-CEA/TRICOM vectors, but also with mice receiving FP-WT vector via the i.v. route. Although it has been reported that the i.v. administration of rF-ß-galactosidase was effective in treating 3-day lung metastases expressing ß-galactosidase in conventional mice (51 , 68) , no comparison of i.v. versus s.c. administration was reported in those studies and it should be noted that much lower doses (106 pfu) were used in those experiments. Additionally, in a separate study, the observation was made that the immune response to antigen administered by the i.v. route could not be augmented by the use of GM-CSF (70) . It is apparent from the data presented here and in other studies that the local administration of recombinant GM-CSF protein (24 , 37 , 59) or the rF vector rF-GM-CSF (38) enhances immune responses to s.c. injected antigen. Whereas the i.v. administration of vaccines may represent a viable alternative to the s.c. route, the use of locally acting cytokines such as GM-CSF cannot be used in those vaccine protocols to enhance the immune response. To our knowledge, the studies reported here are the first comparison of the use of fowlpox vectors by these routes and should discourage the administration of high doses of this vector by the i.v. route.
A previous study has demonstrated that one dose of rV-CEA/TRICOM was superior to one dose of rV-CEA/B7-1 or rV-CEA in inducing T-cell responses (32)
. In the experiments reported here (Fig. 2)
, it was shown that one vaccination of rF-CEA/TRICOM elicits greater T-cell responses than even four vaccinations with either rF-CEA or one or two vaccinations with rF-CEA/B7-1. Moreover, with repeated administration of rF-CEA/TRICOM, CEA-specific T-cell responses as well as CEA-specific antibody responses continued to increase (Figs. 2
and 3
). This is most likely attributable to the fact that avipox viruses, which are replication competent in avian species, are defective for replication in mammalian cells. The CEA and TRICOM transgenes inserted into the avipox vector were done so under the transcriptional control of early pox virus promoters, while most structural viral proteins are under the control of late viral promoters (71
, 72)
. Indeed, in the studies conducted here, antifowlpox virus antibody was induced after each rF vector vaccination (data not shown), but as depicted in the data in Figs. 2
and 3
, there is little if any evidence of virus-neutralizing activity. These results support previous findings that avipox recombinant vectors can be given repeatedly to increase antigen-specific T cell and antibody responses. This was observed in both a murine model as reported here and recently in clinical studies (42)
.
Previous studies have shown that rV viruses can be given no more than one or two times in mice or humans that are vaccinia immune. Several groups have now demonstrated the validity of diversified vaccination protocols where two different immunogens are superior to the repeated use of one immunogen (26
, 29
, 32) . It was unclear, however, whether this diversified vaccination scheme would be necessary with vectors containing multiple costimulatory molecules that are capable of enhancing T-cell responses to far greater levels than vectors devoid of such molecules. Fig. 4
demonstrates that a prime with rV-CEA/TRICOM followed by a boost with rF-CEA/TRICOM was superior to the repeated use of rF-CEA/TRICOM alone. This was demonstrated in both vaccinia naive and vaccinia immune mice. In recent clinical trials (42)
, patients receiving a primary vaccination with rV-CEA and up to eight avipox-CEA booster vaccinations showed increasing precursor frequencies of CEA-specific T cells with each vaccination. These data were obtained using an enzyme-linked immunospot assay in which the cells of patients were only incubated with CEA peptide overnight, to rule out the possibility of artifacts attributable to in vitro stimulation cycles.
Another concept examined in the studies reported here is the use of the cytokine GM-CSF as a biological adjuvant. Recombinant GM-CSF has been used in numerous vaccine protocols in both experimental and clinical studies to demonstrate enhanced T-cell responses to antigens. The vast majority of these studies have administered recombinant GM-CSF on the day of vaccine and for 3 or 4 consecutive days following vaccination. Recent studies have shown that a single administration of rF-GM-CSF enhances APCs and dendritic cells in regional nodes to levels greater than those achieved with four daily administrations of recombinant GM-CSF (38)
. Studies were conducted here to determine whether rF-GM-CSF could induce greater T-cell responses in combination with rF-CEA/TRICOM. The results demonstrate (Fig. 5)
an additive effect in the use of a rF-TRICOM vector, and that of a rF-GM-CSF vector, in inducing systemic antigen-specific T-cell responses. Recombinant GM-CSF was used in the primary vaccination with rV-CEA/TRICOM in the antitumor studies reported here to alleviate the concern that the replication competent rV-CEA/TRICOM would lyse cells infected with rF-GM-CSF and thus limit the paracrine effect of the expressed GM-CSF over the 2 or 3 weeks that fowlpox vectors have been shown to express their transgene. Additional studies will be required, and are in progress, to determine the optimal use of the rF-GM-CSF vector with a range of different types of immunogens, i.e., peptides, proteins, and various vectors.
It should be noted, though, that if rF-GM-CSF is used in conjunction with poxvirus vaccines, it may be more accurate to graph the lymphoproliferative response as a function of 
cpm (experimental cpm - cpm of the media control) rather than cpm. As seen in Fig. 5B
, the FP-WT vector, when admixed with rF-GM-CSF, appears to induce a CEA-specific T-cell proliferative response that is equal to or greater than rF-CEA/TRICOM used alone. Even in the absence of antigen, T cells from FP-WT-vaccinated mice proliferate to some degree if rF-GM-CSF is included in the vaccine. When the background proliferation is subtracted and the data graphed as cpm (inset, B), the response to antigen (CEA) in the proliferation assay is specific for those mice receiving rF-CEA/TRICOM. This phenomenon should thus be considered if in vitro lymphoproliferation is used as a readout for vaccine efficacy where rF-GM-CSF is used.
Low-dose IL-2 was also used in these studies with CEA-transgenic mice because it has been previously shown in several vaccine studies (14
, 22
, 23
, 25)
to enhance T-cell responses. It should be pointed out, however, that the induction of antitumor immunity as depicted in Fig. 7
and the CEA-specific CD4+ and CD8+ responses depicted in Fig. 6
were dependent on inclusion of rF-CEA/TRICOM in the vaccine regimen. No antitumor effect or CEA-specific T-cell responses were observed in mice receiving only control vectors, rF-GM-CSF, and low-dose IL-2. In fact, the survival rates and T-cell responses did not significantly differ from those of the control mice receiving only buffer.
Although the studies reported here demonstrate that the repeated use of rF-CEA/TRICOM is more potent in inducing CEA-specific T-cell responses than the repeated use of rF-CEA or rF-CEA/B7-1, they also demonstrate that vaccine strategies such as the use of diversified prime and boost regimens can also be used along with these vectors to further amplify T-cell responses. Although CEA was used as an antigen in these studies, there is no reason to believe that the strategies reported here cannot be used with other antigens associated with infectious diseases or malignancies. Moreover, it is hoped that the studies reported here will work toward a more rational design of vaccine-based clinical trial strategies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH, 10 Center Drive, Room 8B09, Bethesda, MD 20892. Phone: (301) 496-4343; Fax: (301) 496-2756; E-mail address: js141c{at}nih.gov 
2 The abbreviations used are: TAA, tumor-associated antigen; APC, antigen-presenting cell; TRICOM, triad of costimulatory molecules; rV, recombinant vaccinia; rF, recombinant fowlpox; GM-CSF, granulocyte macrophage colony-stimulating factor; IL-2, interleukin 2; CEA, carcinoembryonic antigen; FP-WT, fowlpox wild type; V-WT, vaccinia WT; pfu, plaque-forming unit; ConA, concanavalin A.
Received 11/27/00. Accepted 4/ 3/01.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 M. G. Cottingham, A. van Maurik, M. Zago, A. T. Newton, R. J. Anderson, M. K. Howard, J. Schneider, and M. A. Skinner Different Levels of Immunogenicity of Two Strains of Fowlpox Virus as Recombinant Vaccine Vectors Eliciting T-Cell Responses in Heterologous Prime-Boost Vaccination Strategies. Clin. Vaccine Immunol., July 1, 2006; 13(7): 747 - 757. [Abstract] [Full Text] [PDF]
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 C. P. Tarassoff, P. M. Arlen, and J. L. Gulley Therapeutic vaccines for prostate cancer. Oncologist, May 1, 2006; 11(5): 451 - 462. [Abstract] [Full Text] [PDF]
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P. M. Arlen, J. L. Gulley, C. Parker, L. Skarupa, M. Pazdur, D. Panicali, P. Beetham, K. Y. Tsang, D. W. Grosenbach, J. Feldman, et al. A Randomized Phase II Study of Concurrent Docetaxel Plus Vaccine Versus Vaccine Alone in Metastatic Androgen-Independent Prostate Cancer Clin. Cancer Res., February 15, 2006; 12(4): 1260 - 1269. [Abstract] [Full Text] [PDF]
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C. Palena, K. A. Foon, D. Panicali, A. G. Yafal, J. Chinsangaram, J. W. Hodge, J. Schlom, and K. Y. Tsang Potential approach to immunotherapy of chronic lymphocytic leukemia (CLL): enhanced immunogenicity of CLL cells via infection with vectors encoding for multiple costimulatory molecules Blood, November 15, 2005; 106(10): 3515 - 3523. [Abstract] [Full Text] [PDF]
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S. Yang, J. W. Hodge, D. W. Grosenbach, and J. Schlom Vaccines with Enhanced Costimulation Maintain High Avidity Memory CTL J. Immunol., September 15, 2005; 175(6): 3715 - 3723. [Abstract] [Full Text] [PDF]
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S. Yang, K.-Y. Tsang, and J. Schlom Induction of Higher-Avidity Human CTLs by Vector-Mediated Enhanced Costimulation of Antigen-Presenting Cells Clin. Cancer Res., August 1, 2005; 11(15): 5603 - 5615. [Abstract] [Full Text] [PDF]
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R. Bos, S. van Duikeren, T. van Hall, P. Kaaijk, R. Taubert, B. Kyewski, L. Klein, C. J.M. Melief, and R. Offringa Expression of a Natural Tumor Antigen by Thymic Epithelial Cells Impairs the Tumor-Protective CD4+ T-Cell Repertoire Cancer Res., July 15, 2005; 65(14): 6443 - 6449. [Abstract] [Full Text] [PDF]
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C. Kudo-Saito, J. Schlom, K. Camphausen, C. N. Coleman, and J. W. Hodge The Requirement of Multimodal Therapy (Vaccine, Local Tumor Radiation, and Reduction of Suppressor Cells) to Eliminate Established Tumors Clin. Cancer Res., June 15, 2005; 11(12): 4533 - 4544. [Abstract] [Full Text] [PDF]
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 D. P. Webster, S. Dunachie, J. M. Vuola, T. Berthoud, S. Keating, S. M. Laidlaw, S. J. McConkey, I. Poulton, L. Andrews, R. F. Andersen, et al. Enhanced T cell-mediated protection against malaria in human challenges by using the recombinant poxviruses FP9 and modified vaccinia virus Ankara PNAS, March 29, 2005; 102(13): 4836 - 4841. [Abstract] [Full Text] [PDF]
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C. Kudo-Saito, J. Schlom, and J. W. Hodge Induction of an Antigen Cascade by Diversified Subcutaneous/Intratumoral Vaccination Is Associated with Antitumor Responses Clin. Cancer Res., March 15, 2005; 11(6): 2416 - 2426. [Abstract] [Full Text] [PDF]
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 J. L. Marshall, J. L. Gulley, P. M. Arlen, P. K. Beetham, K.-Y. Tsang, R. Slack, J. W. Hodge, S. Doren, D. W. Grosenbach, J. Hwang, et al. Phase I Study of Sequential Vaccinations With Fowlpox-CEA(6D)-TRICOM Alone and Sequentially With Vaccinia-CEA(6D)-TRICOM, With and Without Granulocyte-Macrophage Colony-Stimulating Factor, in Patients With Carcinoembryonic Antigen-Expressing Carcinomas J. Clin. Oncol., February 1, 2005; 23(4): 720 - 731. [Abstract] [Full Text] [PDF]
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A. Saha, S. K. Chatterjee, K. A. Foon, F. J. Primus, S. Sreedharan, K. Mohanty, and M. Bhattacharya-Chatterjee Dendritic Cells Pulsed with an Anti-Idiotype Antibody Mimicking Carcinoembryonic Antigen (CEA) Can Reverse Immunological Tolerance to CEA and Induce Antitumor Immunity in CEA Transgenic Mice Cancer Res., July 15, 2004; 64(14): 4995 - 5003. [Abstract] [Full Text] [PDF]
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M. Chakraborty, S. I. Abrams, C. N. Coleman, K. Camphausen, J. Schlom, and J. W. Hodge External Beam Radiation of Tumors Alters Phenotype of Tumor Cells to Render Them Susceptible to Vaccine-Mediated T-Cell Killing Cancer Res., June 15, 2004; 64(12): 4328 - 4337. [Abstract] [Full Text] [PDF]
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H. E. Zeytin, A. C. Patel, C. J. Rogers, D. Canter, S. D. Hursting, J. Schlom, and J. W. Greiner Combination of a Poxvirus-Based Vaccine with a Cyclooxygenase-2 Inhibitor (Celecoxib) Elicits Antitumor Immunity and Long-Term Survival in CEA.Tg/MIN Mice Cancer Res., May 15, 2004; 64(10): 3668 - 3678. [Abstract] [Full Text] [PDF]
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R. J. Anderson, C. M. Hannan, S. C. Gilbert, S. M. Laidlaw, E. G. Sheu, S. Korten, R. Sinden, G. A. Butcher, M. A. Skinner, and A. V. S. Hill Enhanced CD8+ T Cell Immune Responses and Protection Elicited against Plasmodium berghei Malaria by Prime Boost Immunization Regimens Using a Novel Attenuated Fowlpox Virus J. Immunol., March 1, 2004; 172(5): 3094 - 3100. [Abstract] [Full Text] [PDF]
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C. Kudo-Saito, J. Schlom, and J. W. Hodge Intratumoral Vaccination and Diversified Subcutaneous/ Intratumoral Vaccination with Recombinant Poxviruses Encoding a Tumor Antigen and Multiple Costimulatory Molecules Clin. Cancer Res., February 1, 2004; 10(3): 1090 - 1099. [Abstract] [Full Text] [PDF]
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J. W. Hodge, D. J. Poole, W. M. Aarts, A. Gomez Yafal, L. Gritz, and J. Schlom Modified Vaccinia Virus Ankara Recombinants Are as Potent as Vaccinia Recombinants in Diversified Prime and Boost Vaccine Regimens to Elicit Therapeutic Antitumor Responses Cancer Res., November 15, 2003; 63(22): 7942 - 7949. [Abstract] [Full Text] [PDF]
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J. Li, W. Li, S. Liang, D. Cai, M. P. Kieny, L. Jacob, A. Linnenbach, J. W. Abramczuk, H. Bender, K. Sproesser, et al. Recombinant CD63/ME491/Neuroglandular/NKI/C-3 Antigen Inhibits Growth of Established Tumors in Transgenic Mice J. Immunol., September 15, 2003; 171(6): 2922 - 2929. [Abstract] [Full Text] [PDF]
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M. Chakraborty, S. I. Abrams, K. Camphausen, K. Liu, T. Scott, C. N. Coleman, and J. W. Hodge Irradiation of Tumor Cells Up-Regulates Fas and Enhances CTL Lytic Activity and CTL Adoptive Immunotherapy J. Immunol., June 15, 2003; 170(12): 6338 - 6347. [Abstract] [Full Text] [PDF]
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J. W. Hodge, D. W. Grosenbach, W. M. Aarts, D. J. Poole, and J. Schlom Vaccine Therapy of Established Tumors in the Absence of Autoimmunity Clin. Cancer Res., May 1, 2003; 9(5): 1837 - 1849. [Abstract] [Full Text] [PDF]
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U. Pertl, H. Wodrich, J. M. Ruehlmann, S. D. Gillies, H. N. Lode, and R. A. Reisfeld Immunotherapy with a posttranscriptionally modified DNA vaccine induces complete protection against metastatic neuroblastoma Blood, January 15, 2003; 101(2): 649 - 654. [Abstract] [Full Text] [PDF]
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J. W. Greiner, H. Zeytin, M. R. Anver, and J. Schlom Vaccine-based Therapy Directed against Carcinoembryonic Antigen Demonstrates Antitumor Activity on Spontaneous Intestinal Tumors in the Absence of Autoimmunity Cancer Res., December 1, 2002; 62(23): 6944 - 6951. [Abstract] [Full Text] [PDF]
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W. M. Aarts, J. Schlom, and J. W. Hodge Vector-based Vaccine/Cytokine Combination Therapy to Enhance Induction of Immune Responses to a Self-Antigen and Antitumor Activity Cancer Res., October 15, 2002; 62(20): 5770 - 5777. [Abstract] [Full Text] [PDF]
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H. Kobayashi, R. Omiya, M. Ruiz, E. Huarte, P. Sarobe, J. J. Lasarte, M. Herraiz, B. Sangro, J. Prieto, F. Borras-Cuesta, et al. Identification of an Antigenic Epitope for Helper T Lymphocytes from Carcinoembryonic Antigen Clin. Cancer Res., October 1, 2002; 8(10): 3219 - 3225. [Abstract] [Full Text] [PDF]
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E. S. Kass, J. W. Greiner, J. A. Kantor, K. Y. Tsang, F. Guadagni, Z. Chen, B. Clark, R. D. Pascalis, J. Schlom, and C. Van Waes Carcinoembryonic Antigen as a Target for Specific Antitumor Immunotherapy of Head and Neck Cancer Cancer Res., September 1, 2002; 62(17): 5049 - 5057. [Abstract] [Full Text] [PDF]
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J. Schmitz, E. Reali, J. W. Hodge, A. Patel, G. Davis, J. Schlom, and J. W. Greiner Identification of an Interferon-{gamma}-inducible Carcinoembryonic Antigen (CEA) CD8+ T-Cell Epitope, Which Mediates Tumor Killing in CEA Transgenic Mice Cancer Res., September 1, 2002; 62(17): 5058 - 5064. [Abstract] [Full Text] [PDF]
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K. Y. Tsang, M. Zhu, J. Even, J. Gulley, P. Arlen, and J. Schlom The Infection of Human Dendritic Cells with Recombinant Avipox Vectors Expressing a Costimulatory Molecule Transgene (CD80) to Enhance the Activation of Antigen-specific Cytolytic T Cells Cancer Res., October 1, 2001; 61(20): 7568 - 7576. [Abstract] [Full Text] [PDF]
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) or VSV-N (
) peptides. Culture supernatants were assayed for IFN-
, and group 3 by 
in B, proliferation in response to the T-cell mitogen ConA is shown in the inset. Equal numbers of T cells from mice vaccinated as described were cultured with 2.5 µg/ml ConA and naive splenocytes as APC. Proliferation of T cells from all groups in response to ConA was measured by [3H]thymidine incorporation and graphed as cpm. Recombinant CEA/TRICOM and control vectors were given s.c. and used at 108 pfu/mouse/injection. Twenty micrograms of recombinant GM-CSF were injected once a day for 4 consecutive days following the prime vaccination in groups 1 and 2. Recombinant IL-2 (16,000 IU) was given by the i.p. route every 12 h for 4 days following each vaccination in groups 1 and 2. rF-GM-CSF was used at 107 pfu/mouse/injection and was admixed with the fowlpox vectors in groups 1 and 2. Error bars, SDs based on the mean of triplicate wells on 96-well culture plates.
), rF-CEA/B7-1 (
), rF-CEA/TRICOM (•), or HBSS buffer (
) or s.c. route (•) three times at 2-week intervals. Two weeks after the last injection, purified splenic T cells were tested in an in vitro lymphoproliferation assay using naive splenocytes as APC and CEA protein or ovalbumin protein as antigen. Proliferation in response to CEA antigen was measured by [3H]thymidine incorporation and proliferation was graphed as a function of cpm versus protein concentration. Proliferation in response to three i.v. injections of HBSS buffer (A, 
; s.c. route, 
). Control mice were injected with only HBSS buffer (