Recombinant modified vaccinia Ankara primes functionally activated CTL specific for a melanoma tumor antigen epitope in melanoma patients with a high risk of disease recurrence
Article first published online: 13 SEP 2004
Copyright © 2004 Wiley-Liss, Inc.
International Journal of Cancer
Volume 113, Issue 2, pages 259–266, 10 January 2005
How to Cite
Smith, C. L., Dunbar, P. R., Mirza, F., Palmowski, M. J., Shepherd, D., Gilbert, S. C., Coulie, P., Schneider, J., Hoffman, E., Hawkins, R., Harris, A. L. and Cerundolo, V. (2005), Recombinant modified vaccinia Ankara primes functionally activated CTL specific for a melanoma tumor antigen epitope in melanoma patients with a high risk of disease recurrence. Int. J. Cancer, 113: 259–266. doi: 10.1002/ijc.20569
- Issue published online: 8 NOV 2004
- Article first published online: 13 SEP 2004
- Manuscript Accepted: 16 JUN 2004
- Manuscript Received: 4 MAY 2004
- The Ludwig Institute for Cancer Research
- Oxxon Pharmaccines, Ltd.
- Cancer Research UK. Grant Number: C399/A2291
- cytotoxic T lymphocyte (CTL);
- tumor antigen;
Recombinant plasmid DNA and attenuated poxviruses are under development as cancer and infectious disease vaccines. We present the results of a phase I clinical trial of recombinant plasmid DNA and modified vaccinia Ankara (MVA), both encoding 7 melanoma tumor antigen cytotoxic T lymphocyte (CTL) epitopes. HLA-A*0201-positive patients with surgically treated melanoma received either a “prime-boost” DNA/MVA or a homologous MVA-only regimen. Ex vivo tetramer analysis, performed at multiple time points, provided detailed kinetics of vaccine-driven CTL responses specific for the high-affinity melan-A26, 27, 28, 29, 30, 31, 32, 33, 34, 35 analogue epitope. Melan-A26-35-specific CTL were generated in 2/6 patients who received DNA/MVA (detectable only after the first MVA injection) and 4/7 patients who received MVA only. Ex vivo ELISPOT analysis and in vitro proliferation assays confirmed the effector function of these CTL. Responses were seen in smallpox-vaccinated as well as vaccinia-naïve patients, as defined by anti-vaccinia antibody responses demonstrated by ELISA assay. The observations that 1) CTL responses were generated to only 1 of the recombinant epitopes and 2) that the magnitude of these responses (0.029–0.19% CD8+ T cells) was below the levels usually seen in acute viral infections suggest that to ensure high numbers of CTL specific for multiple recombinant epitopes, a deeper understanding of the interplay between CTL responses specific for the viral vector and recombinant epitopes is required.
The characterization of tumor antigens recognized by cytotoxic T lymphocytes (CTL) has led to large numbers of human trials of vaccines designed to stimulate anti-tumor CTL responses. A variety of approaches have been employed, including peptides,1, 2, 3, 4, 5 whole proteins and dendritic cells,6, 7, 8 many of which have shown promising clinical and immunological responses. However, vaccine-driven CTL responses have not always correlated with clinical response. The expression of tumor antigen on tumor cells within an individual is known to be heterogeneous,9 and it has been proposed that this is a result of selection pressure by tumor antigen-specific CTL.10, 11 Vaccination strategies against multiple tumor antigens are therefore aimed at counteracting this tumor escape mechanism. Recently, recombinant gene technology has led to the development of viral and plasmid DNA vaccine delivery systems, and a recombinant “polyepitope” approach, involving a “string” of known tumor antigen epitopes inserted into a vector delivery system, allows multiple epitopes to be delivered simultaneously.
The Poxviridae family of viruses has proven to be particularly suited to the development of recombinant vaccines, and large numbers of animal studies have shown them to be efficient inducers of CTL responses specific for recombinant gene products.12 MVA, a highly attenuated vaccinia virus, incapable of replication in human and most other mammalian cells, has been shown to be capable of inducing strong CTL and antibody responses against recombinant antigens, leading to protective immunity in preclinical animal models.13, 14, 15, 16 Preclinical studies have also demonstrated that some of the most successful vaccination protocols are heterologous “prime-boost” regimens, involving sequential injections of different vectors encoding the same recombinant antigen.17 These regimens are designed to focus the CTL response on the recombinant antigen, which contains the only CTL epitopes shared by the different delivery vectors.18 A “prime-boost” combination of plasmid DNA and MVA has been shown to be particularly effective in animal models.15, 19, 20, 21 Although McConkey et al.5 have recently published results from human trials demonstrating effective generation of anti-malaria T cell responses using DNA/MVA “prime-boost” protocols, evidence of the immunogenicity of recombinant plasmid DNA in humans remains controversial.22, 23, 24
We have engineered 2 recombinant vaccine constructs: plasmid DNA (DNA.Mel3) and MVA (MVA.Mel3), both encoding the same polyepitope string (Mel3) of 7 HLA-A*0201- and HLA-A*01-restricted CTL epitopes from 5 well-defined melanoma tumor antigens (melan-A, NY-ESO-1, MAGE-1, MAGE-3 and tyrosinase),15 (Fig. 1). We have previously shown that each of the epitopes is properly processed and presented to epitope-specific CTL clones.15 We have also demonstrated in HLA-A2-transgenic mice that a heterologous “prime-boost” protocol of DNA.Mel3 and MVA.Mel3 generated up to 100-fold higher CTL expansions than sequential injections of the same vaccine vector.15
To assess the safety and immunogenicity of DNA.Mel3 and MVA.Mel3, we performed a phase I clinical trial in HLA-A*0201-positive patients with resected melanoma and a high risk of disease recurrence. We examined 2 vaccination regimens: a heterologous “prime-boost” DNA/MVA regimen and a homologous MVA-only regimen. We performed detailed immunomonitoring, including ex vivo tetramer and ELISPOT analyses and in vitro proliferation assays of CTL responses specific for the recombinant tumor antigen epitopes in the Mel3 polyepitope string. We also measured antibody responses generated against the MVA viral vector. Our results confirmed the safety of both vaccine constructs and demonstrated that recombinant MVA is capable of eliciting a CTL response specific for the high affinity melan-A26, 27, 28, 29, 30, 31, 32, 33, 34, 35 analogue peptide in 50% of the patients, even in the context of previous vaccination against smallpox. Importantly however, no vaccine-driven CTL responses specific for any of the other epitopes in the Mel3 polyepitope string were demonstrated. These results have important implications for future recombinant polyepitope vaccine design and demonstrate that alternative strategies need to be undertaken to generate broad CTL responses.
Patients and methods
DNA.Mel3 and MVA.Mel3 were manufactured by Qiagen GmbH/DSM Biologics and Impfstoffwerk Dessau-Tornau GmbH (IDT) (Dessau, Germany), respectively, according to cGMP guidelines. DNA.Mel3 was administered at a dose of 1,000 μg intramuscularly, and MVA.Mel3 was administered at 5 × 107 plaque forming units intradermally. The Mel3-encoded epitopes are: tyrosinase1-9, melan-A26–35 analogue (substitution Ala to Leu at residue 2725), tyrosinase369–377,26 MAGE-3168–176, MAGE-3271–279, MAGE-1161–169, NY-ESO-1155–167 and a murine H2-Db restricted influenza virus (flu) nucleoprotein epitope,27 which was included in the construct for preclinical analyses (see Palmowski et al.15 for full insert sequence) (Fig. 1). The tumor antigen epitopes are restricted by HLA-A*0201, except MAGE-3168–176 and MAGE-1161–169, which are HLA-A*01-restricted (Fig. 1).
Treatment was carried out at the Cancer Research UK Medical Oncology Unit, Oxford, U.K. and The Christie Hospital, Manchester, U.K. The protocol was approved by the relevant Local Research Ethics Committees and was carried out according to the declaration of Helsinki. The protocol was also approved by the U.K. Gene Therapy Advisory Committee. All patients gave written, informed consent prior to study entry.
Patients were randomized to receive 2 injections of DNA.Mel3 followed by 2 injections of MVA.Mel3 (Arm A), or 4 injections of MVA.Mel3 (Arm B). Injections were administered every 2 weeks. Randomization was performed to avoid treatment group allocation bias, but the study was not designed to perform statistical comparisons between the 2 treatment groups.
Fourteen patients were enrolled (Table I). Inclusion criteria were the following: HLA-A*0201-positivity, adequate surgery for cutaneous melanoma, a 50% or greater chance of disease recurrence (primary tumor ≥ 4.0 mm thick or ≥ 2.5 mm with ulceration, stage III, or excised skin metastases) and no evidence of metastatic disease on computerized tomography of brain, chest, abdomen and pelvis performed within 4 weeks prior to study entry. Exclusion criteria were the following: surgery, radiotherapy or vaccination within 4 weeks; chemotherapy or immunotherapy within 1 year; immunodeficiency; autoimmune disease and egg allergy. Whilst on study, 3 patients (08, 10 and 11) developed skin nodules that were confirmed to be melanoma on excision after study completion. Patient 01 withdrew from the study early for reasons unrelated to the clinical trial and was not included for immunomonitoring.
|Patient number||Age||Sex||Tumor stage1||Previous treatment||Randomization2||Control peptide3|
|10||63||F||IV (skin metastases)||Surgery||B||CMV|
|1145||56||M||IV (skin metastases)||Surgery||A||CMV|
Safety was assessed by monitoring systemic and local toxicities, which were graded according to the National Cancer Institute Common Toxicity Criteria (NCI CTC) scale. Patients were observed closely for 3 hr after each injection. Two days after each injection an assessment of the local skin reaction was performed. Clinical haematology and biochemistry tests were performed on days 0, 2, 7, 14, 28, 42, 56 and 70.
Blood samples were taken at 16 time points during the 70-day monitoring period. Peripheral Blood Mononuclear Cells (PBMC) were separated, cryopreserved in 10% DMSO and 40% FCS in RPMI and stored in liquid nitrogen. Serum samples were stored at −20°C. For each assay, PBMC or serum from the time points studied were thawed and analyzed concurrently to ensure consistent comparison across time points.
Peptides were synthesized by FMOC chemistry in house, or purchased (Sigma-Genosys, The Woodlands, TX, or Invitrogen, Carlsbad, CA). Tetramers were synthesized as previously described,28 and doses determined by titration against CTL clones and PBMC, including HLA-A*0201-negative PBMC as negative controls.29 The melan-A26–35 tetramer was synthesized using the analogue peptide. In every experiment, relevant CTL clones and HLA-A*0201-negative PBMC were included to validate tetramer staining. Anti-CD8α (PerCP) and anti-CD45R0 (APC) were from BD Biosciences (Mountain View, CA); anti-CD45RA (FITC), anti-CD27 (FITC) and anti-CD28 (APC) were from BD PharMingen, (San Diego, CA).
Ex vivo tetramer analysis.
Thawed PBMC were stained with PE-labeled tetramer and antibodies as described previously.29 Cells were kept on ice without fixation and analyzed on a Becton Dickinson FACScalibur® using CellQuest® software. Small lymphocytes were gated according to forward/side scatter, and dead cells were excluded on the basis of atypical fluorescence. Tetramer+ CD8+ cells were defined as CD8high with a PE-fluorescence equivalent to that of a tetramer-stained clone. A detection threshold for a tetramer+ cell population in PBMC was defined as 100 tetramer+ cells/106 CD8+ cells (0.01% CD8+).4 We defined a positive response to the vaccine as an expansion in the number of melan-A26–35 tetramer+ cells either to > 0.02% CD8+ on 2 consecutive time points if the baseline value was <0.01% CD8+, or to > 3× the baseline value on 2 consecutive time points if it was above 0.01% CD8+ cells. Where replicate analyses were performed, results are given as the arithmetic mean, and the error bars signify ± 1 SD. For phenotypic analysis, profiles of tetramer+ CD8+ cells were compared to whole PBMC for that patient.
The recall assay induces proliferation of peptide-specific CD8+ cells in PBMC samples to increase the frequency of cells detectable with tetramers and validate ex vivo results. PBMC were incubated at 1 × 105 cells/well of a 96-well round-bottomed plate, in 200 μl Iscove's Modified Dulbecco's Medium (IMDM) with 20 μM peptide, for 1 hr. For analysis of melan-A26, 27, 28, 29, 30, 31, 32, 33, 34, 35-specific CTL responses the analogue peptide was used. After washing, 4 × 105 unpulsed PBMC were added to each well in 250 μl IMDM with 5% human serum (I5) and 100 U/ml IL-2 (Chiron, Emeryville, CA). After 7-day culture, cells were stained with appropriate tetramer and PerCP anti-CD8α. Every assay testing recall responses to tumor epitopes also included a viral control peptide recognized by each patient (Table I), which was selected from Influenza (flu) matrix protein58–66, Epstein Barr virus (EBV) BMLF1 lytic protein280–288, and cytomegalovirus (CMV) pp65 lower matrix protein495–503, according to the largest peptide-specific CTL population observed on tetramer staining of a PBMC sample for each patient.
Enzyme-linked immunospot (ELISPOT) assay.
ELISPOT analysis of Interferon-gamma (IFN-γ) secretion (Mabtech, Stockholm, Sweden) was performed as previously described.29 Thawed PBMC were rested overnight, plated in duplicates of 5 × 105 cells/well in I5 with or without 10 μM peptide, and incubated for 36 hr. For analysis of melan-A26–35-specific CTL responses the analogue peptide was used. Each assay included a viral control peptide as in the recall assay. Spots were counted using an automated AID ELISPOT reader (Autoimmun-Diagnostika, Strassberg, Germany). Results were calculated by subtracting the mean background spot count from the mean spot count for each peptide and adjusted to % of total CD8+ cells (determined by flow cytometry). Assays were excluded if counts were > 25 spots per well in the absence of peptide, and a detection threshold was defined as 10 spots per well above no-peptide controls (equivalent to 0.01% CD8+ cells).30
Anti-MVA IgG ELISA.
Serum ELISA for anti-viral IgG antibodies was performed as previously described,31 using 96-well Maxisorp ELISA plates (Nunc, Denmark) coated with 50 μl per well of MVA.Mel3 at 10 μg protein/ml buffer.
Immune responses to the recombinant Mel3 epitopes
Expansion of melan-A26–35-specific CTL.
We and others have shown that HLA-A*0201-positive individuals have a higher frequency of melan-A26–35-specific CTL precursors than for any of the other Mel3 epitopes.29, 30, 32 Therefore, we first analyzed the frequency of melan-A26–35-specific CTL by ex vivo tetramer analysis of PBMC from all 16 study time points for each patient (Fig. 2). This analysis was performed in duplicate (Fig. 2). According to the stringent criteria we set for a positive vaccine-driven CTL response determined by ex vivo tetramer analysis (outlined in the Material and Methods), 6/13 patients developed a melan- A26–35-specific CTL response over the course of the study. Two patients (02 and 08) had received DNA/MVA, and 4 (04, 07, 09 and 10) patients had received MVA only (Fig. 2). The range of peak responses was 0.029–0.19% CD8+ cells.
The large number of immunomonitoring time points in this clinical trial enabled the kinetics of vaccine-driven epitope-specific CTL responses to be visualized in unprecedented detail for a clinical trial (Fig. 2). Expansions in the number of melan-A26–35-specific CTL were seen after 1 or 2 MVA.Mel3 injections. All of the responding patients who received 4 injections of MVA.Mel3 (04, 07, 09 and 10) demonstrated a plateau in the expansion of melan-A26–35-specific CTL after 2 or 3 MVA.Mel3 injections. Responses in both of the responding patients who received the DNA/MVA “prime-boost” regimen (02 and 08), melan-A26–35-specific CTL were detectable ex vivo only after the first MVA.Mel3 injection.
Expanded melan-A26–35-specific CTL have effector phenotype and function.
Phenotypic analysis of melan-A26–35-specific CTL in the prevaccination samples confirmed our previous results that showed that early stage melanoma patients, as well as normal healthy individuals,29, 30 have melan-A26–35-specific CTL with functional and phenotypic markers of a naïve population. In prevaccination samples, tetramer+ melan-A26–35-specific CTL were predominantly CD45RA+/CD45R0−, consistent with a naïve phenotype (Fig. 3a). In contrast, vaccine-driven expanded populations of tetramer+ melan-A26–35-specific CTL also included cells with antigen-experienced CD45RA−/CD45R0+ and CD45RA+/CD45R0+ phenotypes (Fig. 3a).
Ex vivo ELISPOT analysis confirmed that a proportion of vaccine-expanded melan-A26–35-specific CTL were capable of secreting IFN-γ. No responses were detected in prevaccination samples of any of the patients. Responses were demonstrated in postvaccination samples of 3 patients. In Arm A (DNA/MVA), patient 02 showed a response on ELISPOT of 0.08% CD8+ at day 56 and 0.061% CD8+ at day 70 (Fig. 3b). Patient 08 was excluded from analysis because of persistently high background levels. In Arm B (MVA only), the 2 patients with the highest melan-A26–35-specific responses on tetramer analysis also demonstrated a positive response to melan-A26–35 on ELISPOT: patient 04 with 0.021% CD8+ at day 42 (Fig. 3b), and patient 10 with 0.027% CD8+ at day 42 (data not shown). The differences in the magnitude of epitope-specific CTL responses detected on ex vivo ELISPOT and tetramer analyses (approximately 3 times higher with tetramers) are consistent with the previous experience of ourselves and others.4, 29, 30, 33, 34
Recall in vitro proliferation assays confirmed that, for every patient in whom a vaccine-driven expansion of melan-A26–35-specific CTL was detectable on ex vivo tetramer analysis, the expanded CTL were capable of rapidly proliferating in response to melan-A26–35 peptide (Fig. 4 and data not shown). To further address the question of priming by DNA.Mel3, we performed recall assays for the melan-A26–35 peptide at day 28 (after 2 DNA.Mel3 injections, prior to MVA.Mel3) for the 2 patients (02 and 08) in the DNA/MVA Arm who both demonstrated an ex vivo melan-A26–35-specific CTL responses on tetramer analysis after receiving MVA.Mel3. Although ex vivo tetramer analysis of PBMC from time points after DNA.Mel3 and prior to MVA.Mel3 showed no evidence of melan-A26–35-specific CTL, recall assays were performed to exclude the presence of melan-A26–35-specific cells below the limit of detection. For patient 02, there was no difference between prevaccination and day 28 recall responses (Fig. 4), providing no evidence of DNA.Mel3 priming. For patient 08, we observed a prevaccination recall response of 0.04% CD8+ cells after averaging triplicate assays, and the results of triplicate assays performed at day 28 were 0.03%, 0.04% and 1.4% CD8+ cells (data not shown). While these results raise the possibility of priming by DNA.Mel3, they also illustrate the inherent variability associated with in vitro proliferation assays and provide reason for caution when using these assays as the sole readout for immunotherapy trials. This is particularly relevant for epitopes such as melan-A26–35 for which even healthy donors have an atypically elevated baseline level, and for which in vitro priming cannot be excluded.32
Indirect evidence of effector function in vaccine-driven CTL was provided by clinical findings in patient 02 (DNA/MVA), who demonstrated the highest peak of melan-A26–35-specific CTL (0.19% CD8+ T cells). At the same time point that the expansion of melan- A26–35-specific CTL first became detectable by ex vivo tetramer analysis (day 35, 7 days after the first MVA.Mel3 injection), this patient developed redness, itching and crusting of 3 melanocytic naevi, as well as a diffuse macular rash, most prominent around the neck and shoulders. Excision biopsies of 2 of the naevi were performed on day 42. In both cases, histology revealed a lymphocytic infiltrate, consisting of both CD8+ and CD4+ lymphocytes. The rash resolved spontaneously over the following 6 weeks. No vitiligo developed.
Lack of CTL responses to other Mel3 recombinant epitopes.
We screened for the presence of responses to all of the other epitopes in the Mel3 polyepitope string using recall assays and ex vivo ELISPOT analysis (including ELISPOT for HLA-A*01-restricted epitopes in patients 11 and 12). In simultaneous assays, we compared PBMC samples taken prevaccination, postvaccination and at time points at which a peak ex vivo melan-A26–35-specific response was seen on tetramer analysis. Whilst we always detected the presence of CD8+ T cells specific for viral control peptides, neither assay provided any evidence of the presence of CTL specific for the Mel3 epitopes other than melan-A26–35, in any of the samples tested, with only the following exception.
For patient 12 (DNA/MVA), in whom there was no vaccine-driven melan-A26–35-specific CTL response, we demonstrated NY-ESO-1155–167-specific CTL on ex vivo tetramer and ELISPOT analysis, and recall assay (data not shown). There was no difference in any of the assays between pre- and post-vaccination PBMC samples. Ex vivo tetramer analysis performed at each immunomonitoring time point revealed NY-ESO-1155–167-specific CD8+ cells, that were predominantly CD45RA−/CD45R0+, at a frequency of 0.13–0.15% CD8+ across all time points (data not shown).
Anti-viral immune responses
Analysis of serum anti-MVA IgG antibodies demonstrated that all patients developed an increase in the serum titer over the course of the study. Two patterns of response were seen (Fig. 5). Eleven patients developed a rapid increase in the serum titer within 2 weeks of receiving the first dose of MVA.Mel3. The other 2 patients (09 and 11), who reported no previous history of vaccination against smallpox, had prevaccination titers no higher than the negative controls and did not develop an increase in the serum titer until 4 weeks after receiving the first dose of MVA.Mel3. These results are consistent with previously described kinetics of antibody responses, determined by ELISA, in vaccinia-naïve and -nonnaïve volunteers vaccinated with the New York City Board of Health strain of vaccinia.35 Recently, anti-poxviral antibodies determined by ELISA have been shown to correlate closely with the presence of neutralizing antibodies.36
During the clinical trial, a total of 14 DNA.Mel3 and 42 MVA.Mel3 injections were administered. No local reactions developed after any of the DNA.Mel3 injections. All patients developed a mild (NCI CTC Grade 1) local skin reaction 1–3 cm in diameter after each MVA.Mel 3 injection. These reactions resolved within 7 days. Several patients reported mild (NCI CTC Grade 1) flu-like symptoms, characteristically occurring within 24 hr after MVA.Mel3 injections and resolving within the following 24–48 hr. With the exception of the skin reaction in patient 02, described above, no adverse events considered to be likely due to the investigational agents occurred. None of the patients developed abnormalities in complete blood picture, electrolytes or liver function tests.
Our study is the first tumor immunotherapy clinical trial to demonstrate conclusively that recombinant MVA is capable of priming a functional and sustained CTL response specific for a recombinant tumor antigen epitope. Vaccine-driven expansions of CTL specific for the melan-A26–35 epitope were demonstrated in approximately 50% of patients (Fig. 2), and functional analyses demonstrated that these cells were effector cells, as defined by their ability to secrete IFN-γ on ex vivo ELISPOT analysis (Fig. 3b) and to proliferate in vitro in recall assays in the absence of professional APC (Fig. 4).
Although our preclinical studies confirmed that each of the recombinant epitopes in the Mel3 polyepitope string is properly processed and presented to epitope-specific CTL clones,15 no expansion of CTL specific for any of the epitopes other than melan-A26–35 were seen. These results are consistent with our pre-clinical studies of the Mel3 polyepitope string in HLA-A2 transgenic mice, in which we also demonstrated dominance of the melan-A26–35 epitope.15 There are several factors which may have contributed to the dominance of the melan-A26–35 analogue epitope over the other Mel3 recombinant epitopes. Firstly, unlike the other Mel3-encoded epitopes, the melan-A26–35 analogue has a very high peptide binding affinity to HLA-A*0201 molecules.25 Second, healthy individuals as well as melanoma patients have atypically elevated frequencies of melan-A26–35-specific CTL in their peripheral blood.29, 30 We and others have shown that competition between CTL occurs at the surface of the APC and that the simultaneous presentation of different epitopes to a skewed repertoire of primed CTL leads to dominant expansion of a CTL of a limited range of specificities.15, 37, 38 These findings are relevant to the future design of recombinant polyepitope vaccines. To successfully generate CTL responses of multiple specificities, we suggest that epitopes should be separated in the boosting phase, thereby minimizing CTL competition.
Owing to the prevalence of childhood smallpox vaccination, concern has been raised about the possible detrimental effects of preexisting anti-poxviral immune responses on the generation of immune responses against recombinant gene products. Several studies have shown that preexisting immunity against the viral delivery vector impacts negatively on immune responses specific for recombinant gene products.15, 39, 40, 41 Our results showed that preexisting vaccinia-specific immunity, as defined by clinical history and pattern of antibody response, did not prevent a response specific for the recombinant melan-A26–35 epitope: 5 of the 6 melan-A26–35-responding patients, including patient 02 who demonstrated the greatest response, had been vaccinated against smallpox. Furthermore, the 2 vaccinia-naïve patients (patients 09 and 11) did not develop better responses than the smallpox-vaccinated patients.
The detailed kinetics of CTL responses generated in this clinical trial demonstrated that repeated vaccinations with MVA.Mel3 resulted in a plateau of melan-A26–35-specific CTL responses after 2 or 3 injections. This result is consistent with the findings of other groups who have used homologous recombinant viral vaccination strategies.16, 42 Neither this study nor others employing recombinant poxviral vectors have achieved epitope-specific CTL, of the levels that are associated with acute viral infections.43, 44, 45. The likely mechanism for these 2 observations is the generation of poxviral-specific antibody and/or cellular immune responses during the vaccination schedule, either by neutralization of infection or by the generation of immunodominant CTL specific for epitopes encoded within the viral vector itself.46 Therefore, poxviral delivery vectors should best utilized in “prime-boost” vaccination protocols, and consideration should be made to using “smaller” delivery vectors, such as lentivirus, to minimize the impact of vector-specific immune responses. Alternatively, the use of proteins or peptides in combination with adjuvants should be considered: a recent clinical study of repeated injections of peptide and adjuvant has demonstrated continued boosting of CTL responses, reaching levels as high as 8% of CD8+ cells.47, 48
Many preclinical studies have confirmed the ability of recombinant DNA vaccines to prime both humoral and cellular immune responses specific for recombinant gene products in mice and primates.49 Indeed, we confirmed that DNA.Mel3 primes responses specific for the melan-A26–35, tyrosinase369–377 and NY-ESO-1155–167 epitopes in HLA-A2 transgenic mice.15 In contrast, evidence of immunogenicity of plasmid DNA in humans is much more limited.5, 22, 23, 24 Here, we have generated no direct evidence that 2 injections of 1,000 μg DNA.Mel3 are capable of priming an immune response against the recombinant gene products, although the possibility that DNA.Mel3-primed CTL are below the detection limits of the ex vivo assays used in our study, or not recirculating and mainly localized in draining lymph nodes cannot be excluded. Possible contributing factors to the apparent interspecies differences in the immunogenicity of recombinant plasmid DNA are emerging in the literature. They include differences between immune systems, such as the differential expression of Toll-like receptor 9, which recognizes CpG repeat motifs in bacterial DNA.50, 51 Therefore, in addition to addressing such issues as dose, route and method of administration, future clinical trials of DNA vaccination strategies should take these factors into consideration.
In conclusion, our clinical trial demonstrates that recombinant MVA is capable of generating a functionally activated CTL response specific for recombinant gene products. Although recombinant viruses are powerful vaccine delivery vectors, our results highlight several issues relevant to recombinant vaccination protocols using polyepitope strategies and poxviral delivery vectors, which should be considered in future clinical trials.
We thank the Research Nurses and Registrars involved in this trial at the Cancer Research UK Medical Oncology Units in Oxford and Manchester, especially Dr. D. Eaton and D. Selman, and P. Bahl for her technical support. We are grateful to Dr. L.J. Old for his support and critical reading of the article.
- 11An expanded peripheral T cell population to a cytotoxic T lymphocyte (CTL)-defined, melanocyte-specific antigen in metastatic melanoma patients impacts on generation of peptide-specific CTLs but does not overcome tumor escape from immune surveillance in metastatic lesions. J Exp Med 1999; 190: 651–67., , , , , , , , , , .
- 24Safety, tolerability, and lack of antibody responses after administration of a PfCSP DNA malaria vaccine via needle or needle-free jet injection, and comparison of intramuscular and combination intramuscular/intradermal routes. Hum Gene Ther 2002; 13: 1551–60., , , , , , , , , , , , et al.