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Clinical Trials |
1 Cancer Research UK Immunology Group, Paterson Institute for Cancer Research, Christie Hospital, Manchester, United Kingdom; 2 Leiden University Medical Centre, Leiden, the Netherlands; 3 Deutsches Krebsforschungszentrum, Heidelberg, Germany; 4 University of Wales College of Medicine, Cardiff, United Kingdom; 5 Xenova Research Limited, Cambridge, United Kingdom; and 6 Academic Unit of Obstetrics and Gynaecology, St. Mary’s Hospital, Manchester, United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSAnalysis of HPV16 E6-...Analysis of HPV-Specific IgG...RESULTSDISCUSSIONREFERENCES |
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Experimental Design: Twenty-nine women with high-grade AGIN received three i.m. doses of TA-CIN (HPV-16 L2/E6/E7 protein) at four weekly intervals followed by a single dermal scarification of vaccinia HPV-16/18 E6/E7 and were followed up for 12 weeks. Immunity to HPV-16 was assessed by lymphoproliferation, IFN-
enzyme-linked immunospot (ELISPOT), and ELISA.
Results: The patient group significantly responded to TA-CIN and not to the control antigen HPV-6 L2/E7 at all postvaccination time points when compared with baseline responses (P 
0.05). Ten of the patients showed at least a 3-fold increase in TA-CIN-specific proliferation at one or more time points after vaccination. Comparison of stimulation with HPV-16 E6- or E7-GST fusion proteins showed that proliferative responses were biased to HPV-16 E6. This bias was also seen by IFN- ELISPOT using overlapping peptides, with HPV-16 E6- or E7-specific T cells being detected in 9 and 2 patients, respectively. In addition, vaccination resulted in the induction of antibodies against the HPV-16 oncoproteins. Of the 6 clinical responders, 2 patients showed both a proliferative TA-CIN-specific response and an E6-specific IFN- response, whereas 3 other patients displayed E6-specific reactivity only. Stable disease was recorded in 19 patients, 8 of whom showed a concomitant TA-CIN-specific proliferative and/or E6-specific T-cell response. Of the 4 progressors, 2 failed to make a T-cell response and 2 responded by either proliferation or E6 ELISPOT alone.
Conclusions: The prime-boost regimen is immunogenic in AGIN patients (humoral and cellular immunity), but there is no simple relationship between induction of systemic HPV-16-specific immunity and clinical outcome. Other factors that may play a role in the eradication of long-term established AGIN lesions need to be determined to identify the patient group that would benefit from immunotherapy with the vaccines used in this study.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSAnalysis of HPV16 E6-...Analysis of HPV-Specific IgG...RESULTSDISCUSSIONREFERENCES |
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ELISPOT, 5 with a concomitant significant reduction in lesion diameters. Delivery of cure in patients with this and other HPV-associated lesions may depend on viral clearance or at least sustained anti-HPV immunity, and a single vaccination may, therefore, not be enough.
A second vaccine formulation that has been tested in humans consists of a HPV-16 L2E6E7 fusion protein (TA-CIN). It was well tolerated when administered to healthy volunteers and induced antibody and proliferative responses against TA-CIN, as well as IFN- ELISPOT responses to the HPV-16 oncoproteins (7)
. A small study of VIN patients who received three booster vaccinations with TA-CIN between 7 and 15 months after the TA-HPV vaccination (5)
demonstrated HPV-16-specific proliferative T-cell and/or serological activity, but there was no direct correlation between immunological and clinical responses (8)
.
In preclinical studies, a heterologous prime-boost immunization strategy using a HPV-16 L2E6E7 fusion protein (TA-CIN), in combination with TA-HPV, showed enhanced immunogenicity compared with the use of either agent alone (9) . The protocol of TA-CIN followed by TA-HPV was superior to the reciprocal as defined by the induction of T-cell reactivity against the oncoproteins. This may be related to the fact that TA-HPV comprises a multitude of vaccinia-specific T-cell epitopes, which can efficiently compete with the two HPV-16 oncogenes for the attention of the T-cell-mediated immune response. In the case that the immune response is naïve to either of these epitopes, immunity may very well be skewed toward vaccinia-derived epitopes. Immunization with TA-CIN is thus likely to focus the immune response to the oncoproteins, whereas boosting with TA-HPV will increase the magnitude of this oncoprotein-specific T-cell response. Here, we describe the immunogenicity of a similar heterologous prime-boost strategy of three monthly immunizations with TA-CIN, followed by a single boost with TA-HPV in patients with HPV-16-associated AGIN.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSAnalysis of HPV16 E6-...Analysis of HPV-Specific IgG...RESULTSDISCUSSIONREFERENCES |
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ELISPOT responses at this dose; Ref. 7
), followed 4 weeks later by a boost vaccination comprising a single dose (2.5 x 105 plaque-forming units) of TA-HPV by dermal scarification. Women were followed up for 12 weeks after completion of the vaccination schedule. Blood was taken before vaccination (week 0) and at 12, 16, 20, and 24 weeks for assessment of cell-mediated and humoral immunity. Prevaccination samples were limited for 2 patients (404 and 405), and patient 416 baseline sample was taken at week 4. Biopsies were taken at weeks 12 and 24 for histology and HPV status as described previously (5)
. At entry, 26 patients had lesions with HPV-16, 1 had HPV-33 and HPV-58, 1 was HPV high risk but could not be typed, and 1 was HPV negative. Clinical responses were defined as partial if lesion area was reduced by 
50% and a complete response by complete reduction of the lesion with no evidence of disease in the biopsy. Progression was defined as an increase in lesion area by 50%. The clinical findings will be reported elsewhere.7
Immunological Assays
Analysis of HPV-16-Specific T-Cell Proliferation.
Blood samples were collected in monovettes containing citrate and peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation. Cells were cryopreserved in liquid nitrogen until additional use. PBMCs from all time points for each patient were assessed in a slightly modified assay previously described (5)
and as follows. Briefly, cells were thawed and placed into 24-well suspension cell plates (Sarstedt) for a recovery period of 2 h before being seeded in triplicate wells of a 96-well round bottomed microtiter plate (Alpha Laboratories Ltd.) at 2 x 105 cells/well in RPMI 1640 supplemented with 10% human AB serum (Quest Biomedical), 100 µg/ml streptomycin, and 100 IU/ml penicillin (Life Technologies, Inc.). PBMCs alone (medium control) or PBMCs with 50 µg/ml recombinant HPV-16 L2E6E7 protein (TA-CIN; Xenova Research Ltd.), 50 µg/ml recombinant HPV-6 L2E7 protein (Xenova Research Ltd.), 25 µg/ml HPV-16 GST-E6, 25 µg/ml HPV-16 GST-E7, 25 µg/ml glutathione S-transferase (GST) tag protein alone (all proteins purified on glutathione-Sepharose; Ref. 10
), or 7000 units/ml tuberculin purified protein derivative (Evans Vaccines Ltd., Liverpool, United Kingdom) were incubated for 6 days at 37°C. During the final 18 h of culture, 1 µCi/well [3H]thymidine (NEN Life Science Products) was added. The cells were harvested using a Packard 96-well vacuum cell harvester onto Unifilter plates (Packard BioSciences), left to dry overnight, and 30 µl/well of Microscint 20 scintillation fluid (Packard) added. [3H]Thymidine incorporation was measured using a Topcount scintillation counter (Packard). Replicates were within 10%. Results are presented as stimulation index (SI) = the mean number of counts incorporated by antigen-stimulated PBMCs divided by the mean number of counts for PBMCs in medium alone (negative control); SIs for GST-E6 and GST-E7 were calculated using the response to GST as control. A preexisting proliferative T-cell response to HPV-16 L2E6E7 was defined as SI 2. A postvaccination proliferative T-cell response to HPV-16 L2E6E7 was defined as a 3-fold increase in the SI compared with the prevaccination value. Paired Wilcoxon’s signed rank tests were used to test for population differences in the responsiveness to each antigen before and after vaccination with P 0.05 considered statistically significant. The response to TA-HPV boost vaccination (week 12 versus week 16 and so on) was also compared by Wilcoxon’s signed rank tests.
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Analysis of HPV16 E6- and E7-Specific T-Cell Reactivity by IFN- ELISPOT
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSAnalysis of HPV16 E6-...Analysis of HPV-Specific IgG...RESULTSDISCUSSIONREFERENCES |
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-producing HPV-specific T cells were quantified using ELISPOT that was performed as described previously (7
, 11)
. Briefly, PBMCs were thawed, washed, and seeded at a density of 2 x 106 cells/well of a 24-well plate (Costar, Cambridge, MA) in 1 ml of Iscove’s modified Dulbecco’s medium (BioWhittaker, Verviers, Belgium) enriched with 10% human AB serum in the presence or absence of indicated HPV-16 and HPV-18 E6 and E7 peptide pools. As a positive control, PBMCs were cultured in the presence of a memory recall mix, consisting of a mixture of tetanus toxoid (0.75 limus floccolentius/ml final concentration; National Institute of Public Health and Environment, Bilthoven, the Netherlands), Mycobacterium tuberculosis sonicate [2.5 µg/ml; generously donated by Dr. Paul Klatser, Royal Tropical Institute (Amsterdam, the Netherlands)], and Candida albicans (0.005%, HAL Allergenen Laboratory, Haarlem, the Netherlands). The peptides used spanned the HPV-16 and HPV-18 E6 and E7 protein and consisted of 15 E6 and 9 E7 overlapping 22-mer peptides. Peptides were used in pools of 4–5 peptides at a concentration of 5 µg/ml/peptide. The peptides, as indicated by their first and last amino acid in the protein, were used in the following pools: E6-I: 1–22, 11–32, 21–42, 31–52; E6-II: 41–62, 51–72, 61–82, 71–92; E6-III: 81–102, 91–112, 101–122, 111–132; E6-IV: 111–132, 121–142, 131–152, 137–158; E7-I: 1–22, 11–32, 21–42, 31–52; E7-II: 41–62, 51–72, 61–82, 71–92, 77–98 (HPV-18: last peptide 81–105). After 4 days of incubation at 37°C, PBMCs were harvested, washed, and seeded in four replicate wells at a density of 105 cells/well in 100 µl of Iscove’s modified Dulbecco’s medium enriched with 10% FCS in a Multiscreen 96-well plate (Millipore, Etten-Leur, the Netherlands) coated with an IFN- catching antibody (Mabtech AB, Nacka, Sweden). Additional antibody incubations and development of the ELISPOT were performed according to the manufacturer’s instructions (Mabtech). Spots were counted with a fully automated computer-assisted video imaging analysis system (Bio Sys). Specific spots were calculated by subtracting the mean number of spots +2 x SD of the medium only control from the mean number of spots in experimental wells. Antigen-specific T-cell frequencies were considered to be increased compared with nonresponders when specific T-cell frequencies were 1/10,000 (11)
. T-Cell frequencies were considered to be boosted by the vaccine when they were at least 3-fold higher than before vaccination (7)
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Analysis of HPV-Specific IgG Antibodies by ELISA |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSAnalysis of HPV16 E6-...Analysis of HPV-Specific IgG...RESULTSDISCUSSIONREFERENCES |
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0.05 considered statistically significant. |
RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSAnalysis of HPV16 E6-...Analysis of HPV-Specific IgG...RESULTSDISCUSSIONREFERENCES |
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shows an example of such proliferative responses detected in an individual patient. A 3–4-fold increase in TA-CIN-specific proliferation was detected after the three prime vaccinations with TA-CIN. This is not boosted by TA-HPV at week 16 but continues above baseline over the course of the study. The group responses to the HPV antigens are shown as box whisker plots in Fig. 1B
. The patient group as a whole showed significantly increased TA-CIN-specific responses after the three TA-CIN vaccinations, which was sustained on weeks 16–24. There was no significant change in response to the control antigen HPV-6 L2/E7 at any time point (Table 1)
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Examination of the proliferative responses against the two oncoproteins using recombinant HPV-16 GST-E6 or GST-E7 proteins in the proliferation assay showed that group responses were significantly increased only for GST-E6 at weeks 16 (Table 1)
. The ratio of E6 to E7 responses over the trial period supports this bias for E6-specific T-cell proliferation (Fig. 1C)
. The overall magnitude of response, reflected by the SI range and median values, is greater for TA-CIN than E6-GST (Fig. 1)
, implying that the magnitude of the T-cell response reactive with non-E6 or E7-encoded T-cell epitopes is higher. A comparison of proliferative responses at weeks 12 (before TA-HPV) and 16 showed evidence for a booster effect of the TA-HPV vaccination for E6-GST reactivity (P = 0.045) but not for TA-CIN (P = 0.16). All patient lymphocytes showed a proliferative response to tuberculin purified protein derivative, indicating that there was no overt immune suppression in these patients as well as that the samples tested were of good quality.
Vaccine-Induced IFN--Producing T-Cell Reactivity to HPV-16/18 E6 and E7.
IFN- ELISPOT assays were performed with PBMCs provided from 25 patients, taken before vaccination (week 0) and following all vaccinations (week 16 and/or week 20/24). HPV-16-specific T-cell responses were detected in 11 of 25 patients (Table 2)
. Preexisting HPV-16-specific immunity was detected in 3 patients (201, 301, and 412), all of whom showed reactivity to HPV-16 E6 peptides. None of the patients showed HPV-16 E7-specific T cells before vaccination. Vaccine-induced HPV-16 E6-specific T cells were found in 9 of 25 patients after all vaccinations (Table 2)
. All of the patients who displayed at least a 3-fold increase in E6-specific reactivity in the proliferation assays (201, 301, 304, 310, and 311) responded to one or more pools of E6 peptides in the ELISPOT assay. In two patients (301 and 405), HPV-16 E7-specific T cells were enhanced after all vaccinations (Table 2
and Fig. 2
).
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ELISPOT assays for the detection of HPV-16- and HPV-18 E6- and E7-specific T cells, we found that 11 of 25 patients responded to vaccination.
Induction of HPV-16E6- and E7-Specific Antibodies.
Compared with normal control subjects, the patients before vaccination had evidence of significantly higher levels of antibodies to HPV-16 E7 (P = 0.0001), HPV-18 E7 (P = 0.036), and HPV-16 L1 (P = 0.0001) in a nonpaired nonparametric Mann-Whitney U test. Thirteen patients had HPV-16 L1-specific antibodies at baseline greater than the arbitrary cutoff generated from the normal blood donors; these L1 antibody levels remained stable over the course of the study. Fig. 3
shows box-whisker plots of group serological responses to HPV-16 E6 and E7 after vaccination and statistical analysis. Table 3
shows significant group responses at all time points compared with baseline for both HPV-16 E6 and E7 but not L1. No significant changes were seen in serological responses to HPV-18 E6 or E7 after vaccination. As defined by a 3-fold increase from baseline and being above the normal cutoff level, 14 of 24 patients tested were categorized as showing positive IgG responses to HPV-16 E7, 1 of whom also had positive responses to HPV-16 E6 (Table 4)
. The responses to HPV-16 E6 or E7 were transient, with levels peaking at week 16 and declining by weeks 20 and 24, consistent with the single booster vaccination with E6 and E7 encoding TA-HPV at week 12.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSAnalysis of HPV16 E6-...Analysis of HPV-Specific IgG...RESULTSDISCUSSIONREFERENCES |
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Strong proliferative TA-CIN-specific responses were found in the AGIN patients after the vaccinations with TA-CIN, and these are of similar magnitude as those found in TA-CIN vaccinated healthy subjects (7) . The higher level of TA-CIN-specific proliferation seen compared with that detected with E6 and the absence of E7-specific T-cell proliferation might be explained by a strong response to HPV-16 L2 or an epitope derivative from the L2/E6/E7 fusion (and absent from the HPV-6 L2E7 control). A preponderance of L2 directed T-cell reactivity may also explain the lack of detection of booster activity by injection with TA-HPV encoding the E6 and E7 oncoprotein only when tested in a proliferation assay with TA-CIN as stimulating antigen. Interestingly, those patients with the lowest preexisting TA-CIN-specific response were most likely to respond vigorously upon vaccination with TA-CIN. This might be indicative of a preexisting response masking vaccination-induced changes, with respect to the quality or type of T-cell response, not measured by the proliferation assays. Therefore, vaccine-induced proliferative responses may only be detected in those patients with no preexisting proliferative responses. Indeed, none of 5 patients in whom an E6-specific vaccine-induced proliferative response was detected had evidence of a preexisting E6 response. These observations point to some limitations of the TA-CIN proliferation assay.
In some of the cases, a nonprotective type of immunity (e.g., Th2, Treg, or nonpolarized) preexisting T-cell response may exist or the E6- and E7-specific T cells could have been anergized through the presentation of the two oncogenes to the immune system in a noninflammatory context, as was found in a HPV-16 mouse model (13)
. The long period over which the E6 and E7 proteins are presented to the immune system in the noninflammatory context of AGIN compared with the lack of L2 production in these lesions may hamper the induction of a strong type 1 T-cell reactivity against the two oncogenes when compared with L2. Indeed, the frequency of HPV-16 E6-specific IFN--T-cell responses were found to be greater in patients with a preexisting Th1 type E6-response, whereas in the other patients, including those in which a vaccine-induced proliferative response was detected, a more modest response was induced (Ref. 6
and this study).
Although priming of E6-specific T-cell reactivity is achieved by vaccination with TA-CIN (Ref. 7
and this study), the major increase in E6-specific T-cell reactivity is due to the booster vaccination with TA-HPV. Analysis of the proliferative group responses revealed that the E6-specific T-cell responses were significantly increased at week 16, which is 4 weeks after vaccination with TA-HPV. Furthermore, our previous analysis of T-cell reactivity in high-grade VIN patients also revealed that vaccination with TA-HPV enhanced especially the numbers of IFN--producing E6-specific T cells (6)
. Such HPV-16 E6-specific T-cell immunity is frequently detected in healthy subjects, and this is supportive of a role in protection against persistent HPV infection and associated development of malignancies (14)
. The fact that the heterologous prime-boost protocol increases the numbers of these effectors should therefore offer potential therapeutic value in some of the high-grade VIN patients. To describe the immunization regimen as prime-boost in this therapeutic approach to treatment of patients with lesions likely containing the HPV-16 proteins used in the vaccine formulations may not be entirely appropriate. However, overall, the protocol is capable of boosting existing T-cell responses from a low level as well as inducing de novo recognition of different epitopes.
The serological data emphasize the patient pre-exposure to HPV-16 infection. There are significant levels of antibodies to HPV-16 E7 and L1 detectable before vaccination, and after the immunization protocol, there are transient increased responses to both E7 and E6 but not L1. The role of these antibodies in the control of HPV lesions is not known and they may simply reflect the generation of T-helper cells, which might also have a positive or negative influence in the lesions through cytokine release.
Clinically, we observed objective responses in 6 patients and 19 had stable disease, and in 4 patients, there was evidence of progression.7
Table 4
summarizes these and the immunological responses. Of the 6 clinical responders, 2 patients showed both a proliferative TA-CIN-specific response and an E6-specific IFN- response, whereas 3 other patients displayed E6 reactivity only. Stable disease was recorded in 19 patients, 8 of whom showed a concomitant TA-CIN-specific proliferative and/or E6-specific T-cell response, whereas in the other 11 patients, no vaccine-induced response could be detected. Of the 4 progressors, 2 failed to make a T-cell response and 2 responded by either proliferation or E6 ELISPOT alone. There is no statistically demonstrable association between clinical response and immunological response measured by either TA-CIN proliferation or ELISPOT or anti-E7 antibodies (odd ratios of 0.36, 0.11, and 0.16, respectively). However, such individual immune responses of patients were defined by 3-fold increases in activity from baseline and are thus a reflection of the magnitude of measurable vaccine induced change rather than inherent HPV immunity. Focusing on combinations of immunological activity, outcome of vaccination in patients and other factors may give another perspective. Our previous analysis of vaccinated VIN patients showed that clinical responsiveness to treatment was dependent on the presence of lesion-associated CD4+, CD8+, and CD1a+ immune cells (5)
, suggesting that not only the numbers of systemically present HPV-16-specific T cells are important but also their capacity to reach the target site. The patients treated in this study have premalignant HPV-associated lesions, which can show evidence of altered HLA class I expression (15)
, thereby facilitating immune escape from any CTL generated by the vaccination. In the design of new trials, local immune infiltration and HLA lesion expression will be important factors to consider in relation to outcome. Preselection of high-grade VIN patients with HLA class I-positive lesions displaying T-cell infiltrate before vaccination may identify a group of subjects who benefits the most from vaccination.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: L. Smyth and M. van Poelgeest contributed equally to this article.
Requests for reprints: Sjoerd H. van der Burg, Department of Immunohematology and Blood Transfusion, Building 1, E3-Q, Leiden University Medical Center, P. O. Box 9600, 2300 RC Leiden, the Netherlands. Phone: 31-71-5266849; Fax: 31-71-5216751; E-mail: shvdburg{at}lumc.nl
7 H. Kitchener, A. Tristram, E. J. Davidson, A. Tomlinson, J. Dobson, P. Baldwin, J. Sterling, and A. Fiander. The clinical effects of a multicentre trial of a prime boost vaccination strategy in women with high grade anogenital intraepithelial neoplasia, submitted for publication. 
Received 12/10/03; revised 1/24/04; accepted 1/30/04.
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REFERENCES |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSAnalysis of HPV16 E6-...Analysis of HPV-Specific IgG...RESULTSDISCUSSIONREFERENCES |
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