Development of a replication‐deficient adenoviral vector‐based vaccine candidate for the interception of HPV16‐ and HPV18‐induced infections and disease

High‐risk Human papilloma virus (HPV) types are the causative agents of cervical cancer and several other anogenital malignancies. The viral proteins expressed in the (pre)malignant cells are considered ideal targets for immunological intervention. Many approaches have been evaluated for this purpose, mostly aiming at the induction of HPV16 E7‐ and/or E6‐specific cellular immunogenicity. As clinical success has so far been limited, novel approaches are required. We describe the development and pre‐clinical testing of a vaccine candidate consisting of replication‐deficient adenovirus type 26 and 35 based vectors for the interception of HPV16‐ and HPV18‐related disease. We developed HPV16‐ and HPV18‐specific antigens consisting of fusion proteins of E2, E6 and E7. The vaccine will be suitable for every disease stage, from incident and persistent infections where E2 is predominantly expressed up to late stages where E6 and E7 expression are upregulated. Importantly E6 and E7 are present as reordered fragments to abrogate the transforming activity of these two proteins. Loss of transforming activity was demonstrated in different in vitro models. Robust T‐cell immunogenicity was induced upon immunization of mice with the vaccine candidate. Finally, the developed vaccine vectors showed considerable therapeutic efficacy in the TC‐1 mouse model. The absence of transforming activity of the antigens and the favorable immunogenicity profile of the adenovirus based vectors along with the fact that these vectors can be readily produced on a large scale makes this approach attractive for clinical evaluation.     

Discovery of chemotherapy‐associated ovarian cancer antigens by interrogating memory T cells

According to the immunogenic cell death hypothesis, clinical chemotherapy treatments may result in CD8⁺ and CD4⁺ T‐cell responses against tumor cells. To discover chemotherapy‐associated antigens (CAAs), T cells derived from ovarian cancer (OC) patients (who had been treated with appropriate chemotherapy protocols) were interrogated with proteins isolated from primary OC cells. We screened for immunogenicity using two‐dimensional electrophoresis gel‐eluted OC proteins. Only the selected immunogenic antigens were molecularly characterized by mass‐spectrometry‐based analysis. Memory T cells that recognized antigens associated with apoptotic (but not live) OC cells were correlated with prolonged survival in response to chemotherapy, supporting the model of chemotherapy‐induced apoptosis as an adjuvant of anti‐tumor immunity. The strength of both memory CD4⁺ and CD8⁺ T cells producing either IFN‐γ or IL‐17 in response to apoptotic OC antigens was also significantly greater in Responders to chemotherapy than in nonresponders. Immunogenicity of some of these antigens was confirmed using recombinant proteins in an independent set of patients. The T‐cell interrogation system represents a strategy of reverse tumor immunology that proposes to identify CAAs, which may then be validated as possible prognostic tumor biomarkers or cancer vaccines.     

Enhancement of DNA vaccine potency by antigen linkage to IFN‐γ‐inducible protein‐10

DNA vaccines have emerged as an attractive approach to generate antigen‐specific T‐cell immune response. Nevertheless, the potency of DNA vaccines still needs to be improved for cancer immunotherapy. In this study, we explored whether functional linkage of a Th1‐polarizing chemokine, IP‐10, to a model tumor antigen, human papillomavirus type 16 (HPV‐16) E7, enhanced DNA vaccine potency. IP‐10 linkage changed the location of E7 from the nucleus to the endoplasmic reticulum and led to the secretion of functionally chemoattractive chimeric IP‐10/E7 protein. In addition, this linkage drastically enhanced the endogenous processing of E7 antigen through MHC class I. More importantly, we found that C57BL/6 mice intradermally vaccinated with IP‐10/E7 DNA exhibited a dramatic increase in the number of E7‐specific CD4⁺ Th1 T‐cells and CD8⁺ T‐cells and, consequently, were strongly resistant over the long term to E7‐expressing tumors compared to mice vaccinated with wild‐type E7 DNA. Thus, because of the increase in tumor antigen‐specific T‐cell immune responses obtained through both enhanced antigen presentation and chemoattraction, vaccination with DNA encoding IP‐10 linked to a tumor antigen holds great promise for treating tumors.     

CD169‐positive sinus macrophages in the lymph nodes determine bladder cancer prognosis

CD169⁺ macrophages are suggested to play a pivotal role in establishing anti‐tumor immunity. They capture dead tumor cell‐associated antigens and transfer their information to lymphocsytes, including CD8⁺ T cells, which is important for successful tumor suppression. This study aimed to determine the prognostic significance of CD169⁺ macrophages residing in the tumor‐draining lymph nodes from cases of bladder cancer. In this retrospective study, 44 bladder cancer patients who received radical cystectomy were examined. The abundance of CD169⁺ macrophages in the regional lymph nodes and the number of CD8⁺ T cells in the primary tumor were investigated by immunohistochemistry. A CD169 score was calculated based on the intensity of CD169 staining and the proportion of CD169⁺ macrophages, and the scores were compared to the patients’ clinicopathological parameters. A high CD169 score was significantly associated with low T stage and with a high number of CD8⁺ T cells infiltrating into the tumor. The group with high CD169 expression had significantly longer cancer‐specific survival than the group with low CD169 expression (5‐year cancer‐specific survival rate: 83.3% vs 31.3%). In a multivariate analysis, the CD169 score was identified as a strong and independent favorable prognostic factor for cancer‐specific survival. Our findings suggest that CD169⁺ macrophages in the lymph nodes enhance anti‐tumor immunity by expanding CD8⁺ T cells in bladder cancer. The CD169 score may serve as a novel marker for the evaluation of bladder cancer prognosis.     

Identification of a naturally processed HLA-A*0201 HPV18 E7 T cell epitope by tumor cell mediated in vitro vaccination

Immunotherapy of HPV-associated disease such as cervical cancer is moving from preclinical investigation to clinical trials. The viral oncoproteins E6 and E7 are ideal target antigens because their expression is mandatory in HPV-transformed tumor cells. T cells are the most important effector cells for therapeutic vaccination strategies. Therefore, the identification and characterization of HPV E6 and E7 T cell epitopes is necessary. Methods to date rely on screening for immunogenicity of peptides predicted by algorithms. Presentation of the identified peptides on tumor cells, however, needs to be confirmed. In our study, we have improved the method to identify peptide epitopes of HPV18 E7 that are actually presented by tumor cells. We induced allogeneic T-cell lines by stimulation with HPV18-positive, CD80 and HLA-A*0201 transfected cervical cancer cells. Sensitized T cells were probed against an array of a HPV18 E7 20mer peptide-library. We found specific reactivity to one of the 20mer peptides. This sequence was then screened via algorithms for putative epitopes. One putative HLA-A2 restricted epitope was confirmed to bind to HLA-A2, to be immunogenic and to induce IFN gamma-release in ELISpot assays. Epitope-specific T cells were cytolytic toward autologous peptide pulsed targets and HPV18 transformed tumor cells. The identification of epitope-specific T cells in tumor infiltrating lymphocytes of a HPV18-positive HLA-matched cervical cancer patient suggests an in vivo relevance of the identified epitope. We suggest that our approach is advantageous over conventional methods, because it yields candidate peptides that are relevant CTL epitopes that are expressed, processed and presented by tumor cells.     

Blood monocytes sample MelanA/MART1 antigen for long‐lasting cross‐presentation to CD8+ T cells after differentiation into dendritic cells

Human blood monocytes are very potent to take up antigens. Like macrophages in tissue, they efficiently degrade exogenous protein and are less efficient than dendritic cells (DCs) at cross‐presenting antigens to CD8⁺ T cells. Although it is generally accepted that DCs take up tissue antigens and then migrate to lymph nodes to prime T cells, the mechanisms of presentation of antigens taken up by monocytes are poorly documented so far. In the present work, we show that monocytes loaded in vitro with MelanA long peptides retain the capacity to stimulate antigen‐specific CD8⁺ T cell clones after 5 days of differentiation into monocytes‐derived dendritic cells (MoDCs). Tagged‐long peptides can be visualized in electron‐dense endocytic compartments distinct from lysosomes, suggesting that antigens can be protected from degradation for extended periods of time. To address the pathophysiological relevance of these findings, we screened blood monocytes from 18 metastatic melanoma patients and found that CD14⁺ monocytes from two patients effectively activate a MelanA‐specific CD8 T cell clone after in vitro differentiation into MoDCs. This in vivo sampling of tumor antigen by circulating monocytes might alter the tumor‐specific immune response and should be taken into account for cancer immunotherapy.     

A novel RNA‐based adjuvant combines strong immunostimulatory capacities with a favorable safety profile

Protein‐ and peptide‐based tumor vaccines depend on strong adjuvants to induce potent immune responses. Here, we demonstrated that a recently developed novel adjuvant based on a non‐coding, long‐chain RNA molecule, termed RNAdjuvant^®, profoundly increased immunogenicity of both antigen formats. RNAdjuvant^® induced balanced, long‐lasting immune responses that resulted in a strong anti‐tumor activity. A direct comparison to Poly(I:C) showed superior efficacy of our adjuvant to enhance antigen‐specific multifunctional CD8⁺ T‐cell responses and mediate anti‐tumor responses induced by peptide derived from HPV‐16 E7 protein in the syngeneic TC‐1 tumor, a murine model of human HPV‐induced cervical cancer. Moreover, the adjuvant was able to induce functional memory responses that mediated complete tumor remission. Despite its remarkable immunostimulatory activity, our RNA‐based adjuvant exhibited an excellent pre‐clinical safety profile. It acted only locally at the injection site where it elicited a transient but strong up‐regulation of pro‐inflammatory and anti‐viral cytokines as well as cytoplasmic RNA sensors without systemic cytokine release. This was followed by the activation of immune cells in the draining lymph nodes. Our data indicate that our RNA‐based adjuvant is a safe and potent immunostimulator that may profoundly improve the efficacy of a variety of cancer vaccines.     

Inhibitors of differentiation‐1 promotes nitrosopyrrolidine‐induced transformation of HPV 16‐immortalized cervical epithelial cell

Our previous study implied a correlation between inhibitors of differentiation‐1 (Id‐1) and cervical cancer development. However, how Id‐1 contributes to cervical carcinogenesis is unknown. In the present study, we used an in vitro transformation model to investigate the role of Id‐1 in the transformation of cervical cells. Human papillomavirus (HPV)‐immortalized cervical epithelial cells (H8) were successfully transformed by exposure to the carcinogen N‐nitrosopyrrolidine (NPYR). The expression of both Id‐1 RNA and protein was significantly increased in transformed H8 cells, suggesting a possible role of Id‐1 in cervical cell transformation. Ectopic expression of Id‐1 in H8 cells potentiated NPYR‐induced cell transformation. In contrast, silencing of Id‐1 suppressed NPYR‐induced H8 cell transformation. In addition, the expression of HPV E6 and E7 oncoproteins was upregulated while that of the tumor suppressors p53 and pRb was suppressed after H8 cell transformation. Our results suggest that Id‐1 plays an oncogenic role in HPV‐related cervical carcinogenesis, which sheds light on cervical cancer development mechanisms and implies that Id‐1 is a potential target for cervical cancer prevention and therapy.     

Alterations of T‐cell surface markers in older women with persistent human papillomavirus infection

We previously reported decreased lymphocyte proliferative responses among older women with persistent human papillomavirus (HPV) infection. To characterize the phenotype of peripheral lymphocytes associated with persistent HPV infection, we evaluated the expression of different cell surface markers in peripheral blood mononuclear cells (PBMCs) from a case–control study within a 10,049 woman population‐based cohort study in Guanacaste, Costa Rica. Women in the cohort aged 46–74 and with HPV results at their 5th year anniversary visit were considered, and all women (n = 87) with persistent HPV infections, all women (n = 196) with transient HPV infections and a random sample of HPV DNA‐negative women (n = 261) frequency‐matched to cases on age were selected for this study. A median of 3 years after the case–control matching visit, cervical cells were collected for liquid‐based cytology and repeat HPV DNA genotyping. Blood was obtained from which PBMCs were extracted and cryopreserved for immunological phenotyping via flow cytometry. Significant increases in risk of HPV persistence were observed for 3 marker subsets indicative of immune cell activation/differentiation. Relative risk estimates were 5.4 (95% CI = 2.2–13.3) for CD69⁺CD4⁺, 2.6 (95% CI = 1.2–5.9) for HLADR⁺CD3⁺CD4⁺ and 2.3 (95% CI = 1.1–4.7) for CD45RO⁺CD27^?CD8⁺. A significant decrease in HPV persistence was observed for a subset marker indicative of an immature, undifferentiated memory state CD45RO⁺CD27⁺CD4⁺ (OR = 0.36; 95% CI = 0.17–0.76). Adjustment for these markers only partially explained the previously reported association between decreased lymphoproliferative responses and persistent HPV infection. Whether phenotypic alterations observed predispose to HPV persistence or result from it should be the focus of future studies.     

Soluble γc receptor attenuates anti‐tumor responses of CD8+ T cells in T cell immunotherapy

Previous studies have shown that soluble common γ‐chain (sγc) modulates CD4⁺ T cell immunity with antagonistic functions in γc cytokine signaling. However, the role of sγc in functional properties of effector CD8⁺ T cells has not been fully defined. In this study, we report a new mechanism by which the anti‐tumor activity of mouse CD8⁺ T cells is suppressed in sγc of their own producing. While sγc significantly inhibits cytotoxicity of CD8⁺ T cells, blocking sγc production by genetic modification leads to potentiated effector function of CD8⁺ T cells, establishing persistent CD8⁺ T cells. This is due to the modulation of IL‐2 and IL‐15 signaling, which is required for expansion and survival of CD8⁺ T cells as well as for optimal cytotoxic activity. More efficient management of tumor growth was achieved by an adoptive transfer of sγc‐deficient CD8⁺ T cells than that of wild‐type or sγc‐overexpressing CD8⁺ T cells. Blocking of IL‐2 and IL‐15 signaling by sγc attenuates the capacity of CD8⁺ T cells to mount an optimal response to the tumor, with both quantitative and qualitative effects on antigen‐specific CD8⁺ T cells. These results could have a critical implication for the generation and survival of optimal effector T cells for adoptive immunotherapy of cancer.     

MHC II lung cancer vaccines prime and boost tumor‐specific CD4+ T cells that cross‐react with multiple histologic subtypes of nonsmall cell lung cancer cells

Nonsmall cell lung cancer (NSCLC) is the major cause of lung cancer‐related deaths in the United States. We are developing cell‐based vaccines as a new approach for the treatment of NSCLC. NSCLC is broadly divided into 3 histologic subtypes: adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Since these subtypes are derived from the same progenitor cells, we hypothesized that they share common tumor antigens, and vaccines that induce immune reactivity against 1 subtype may also induce immunity against other subtypes. Our vaccine strategy has focused on activating tumor‐specific CD4⁺ T cells, a population of lymphocytes that facilitates the optimal activation of effector and memory cytotoxic CD8⁺ T cells. We now report that our NSCLC MHC II vaccines prepared from adeno, squamous or large cell carcinomas each activate CD4⁺ T cells that cross‐react with the other NSCLC subtypes and do not react with HLA‐DR‐matched normal lung fibroblasts or other HLA‐DR‐matched nonlung tumor cells. Using MHC II NSCLC vaccines expressing the DR1, DR4, DR7 or DR15 alleles, we also demonstrate that antigens shared among the different subtypes are presented by multiple HLA‐DR alleles. Therefore, MHC II NSCLC vaccines expressing a single HLA‐DR allele activate NSCLC‐specific CD4⁺ T cells that react with the 3 major classes of NSCLC, and the antigens recognized by the activated T cells are presented by several common HLA‐DR alleles, suggesting that the MHC II NSCLC vaccines are potential immunotherapeutics for a range of NSCLC patients.     

Human papillomavirus (HPV) type 16-specific CD8+ T cell responses in women with high grade vulvar intraepithelial neoplasia

Human papillomavirus (HPV)-associated vulvar intraepithelial neoplasia (VIN) has serious sequelae for the sufferer. Current treatments are associated with poor response and high relapse rates. The development of HPV-specific T cell immunotherapies offers a new approach to treatment. This will require a detailed understanding of the spectrum of T cell responses induced by HPV antigens, and how effectively viral antigens can be accessed by the immune system. We have investigated the frequency and spectrum of HPV16-specific CD8+ T cell responses to three HPV16 antigens in 9 women with high grade VIN (VIN3). CD4-depleted populations of responder cells were screened against overlapping 30-35mer peptides covering the sequences of HPV16 E6, E7 and E4 using ELISPOT assays of IFN-gamma release. We demonstrated CD8+ T cell reactivity to one or more of the proteins in 6 of 9 patient samples. All 6 of these responders recognised peptides covering the E7 protein, 3 of 9 women responded to E6 peptides, but no reactivity was seen to E4. Our results suggest that HPV16-specific cytotoxic T cells (CTLs) are relatively common in women with persistent VIN3. The HPV-specific CTL response, however, seems to be ineffective. There is some evidence that there are problems associated with the processing and presentation of HPV antigens by the infected vulvar epithelium. It will be crucial to address this in the design of any T cell based therapy for HPV-associated VIN and vulval cancer.     

Induction of CD4+ and CD8+ anti‐tumor effector T cell responses by bacteria mediated tumor therapy

Facultative anaerobic bacteria like E. coli can colonize solid tumors often resulting in tumor growth retardation or even clearance. Little mechanistic knowledge is available for this phenomenon which is however crucial for optimization and further implementation in the clinic. Here, we show that intravenous injections with E. coli TOP10 can induce clearance of CT26 tumors in BALB/c mice. Importantly, re‐challenging mice which had cleared tumors showed that clearance was due to a specific immune reaction. Accordingly, lymphopenic mice never showed tumor clearance after infection. Depletion experiments revealed that during induction phase, CD8⁺ T cells are the sole effectors responsible for tumor clearance while in the memory phase CD8⁺ and CD4⁺ T cells were involved. This was confirmed by adoptive transfer. CD4⁺ and CD8⁺ T cells could reject newly set tumors while CD8⁺ T cells could even reject established tumors. Detailed analysis of adoptively transferred CD4⁺ T cells during tumor challenge revealed expression of granzyme B, FasL, TNF‐α and IFN‐γ in such T cells that might be involved in the anti‐tumor activity. Our findings should pave the way for further optimization steps of this promising therapy.     

Cervical cancer cell‐derived interleukin‐6 impairs CCR7‐dependent migration of MMP‐9‐expressing dendritic cells

Cervical carcinogenesis is a consequence of persistent infection with high‐risk human papillomaviruses (HPVs). Recent studies indicate that HPV‐transformed cells actively instruct their microenvironment to promote carcinogenesis. Here, we demonstrate that cervical cancer cells activate monocytes to produce their own CCL2 for further monocyte recruitment and reprogram their function during differentiation and maturation to dendritic cells (DCs). Our data show that cervical cancer cells suppress the induction of the chemokine receptor CCR7 in phenotypically mature DCs and impair their migration toward a lymph node homing chemokine, required to initiate adaptive immune responses. We confirmed the presence of CD83⁺CCR7^low DCs in cancer biopsies. The second factor essential for DC migration, matrix‐metalloproteinase MMP‐9, which also has vasculogenic and protumorigenic properties, is not suppressed but upregulated in immature as well as mature DCs. We identified interleukin‐6 (IL‐6) as a crucial cervical cancer cell‐derived mediator and nuclear factor kappaB (NF‐κB) as the central signaling pathway targeted in DCs. Anti‐IL‐6 antibodies reverted not only NF‐κB inhibition and restored CCR7‐dependent migration but also blocked MMP‐9 induction. This is the first report demonstrating the dissociation of CCR7 and MMP‐9 expression in phenotypically mature CD83⁺ DCs by cancer cells. Our results show that cervical cancer cells actively shape the local microenvironment. They induce the accumulation of myeloid cells and skew their function from immune activation to local production of protumorigenic MMP‐9. Neutralizing anti‐IL‐6 antibodies can counteract this functional dysbalance and should therefore be considered for adjuvant cervical cancer therapy.     

Identification of novel immunogenic human leukocyte antigen-A 2402-binding epitopes of human papillomavirus type 16 E7 for immunotherapy against human cervical cancer

BACKGROUND:

A study was undertaken to identify new immunogenic human leukocyte antigen (HLA)‐A*2402‐restricted epitopes from human papillomavirus (HPV) type 16 E7 protein for immunotherapy against cervical cancer.

METHODS:

Synthetic overlapping peptides were screened by measuring the frequency of CD8⁺ cytotoxic T lymphocytes (CTLs) producing intracellular interferon‐γ (IFN‐γ) using flow cytometry and were validated in SiHa cells with a Cr release cytotoxicity assay. In vivo antitumor effects of peptide‐sensitized peripheral blood mononuclear cells (PBMCs) and isolated CD8⁺ CTLs were evaluated using BALB/c nude mice with SiHa cell xenotransplants.

RESULTS:

Among 14 overlapping 15‐amino acid peptides, E7_61‐75(CDSTLRLCVQSTHVD) and E7_67‐81(LCVQSTHVDIRTLED) induced significantly higher IFN‐γ production (P < .05) and showed higher in vitro cytotoxicity against SiHa cells than did cells sensitized with the negative control. To determine the exact HLA‐A*2402‐restricted epitopes, a total of 25 overlapping 9‐ or 10‐amino acid peptides spanning E7_61‐75 and E7_67‐81 were synthesized. E7_61‐69(CDSTLRLCV) and E7_67‐76(LCVQSTHVDI) induced significantly greater IFN‐γ production as well as increased in vitro cytotoxicity against SiHa cells compared with those of other peptides and the negative control (P < .01), and the antitumor effects of these peptide‐sensitized PBMCs were induced by CD8⁺ CTLs. E7_61‐69‐sensitized and E7_67‐76‐sensitized PBMCs and isolated CD8⁺ CTLs showed a much greater suppression of tumor growth in vivo compared with that of control groups treated with PBS (P < .01). The authors also confirmed the synergistic antitumor effect of cisplatin followed by E7_67‐76‐sensitized PBMCs in vivo.

CONCLUSIONS:

E7_61‐69 and E7_67‐76 were identified as novel HPV type 16 E7 epitopes for HLA‐A*2402, which could be used for immunotherapy against cervical cancer. Cancer 2012. © 2011 American Cancer Society.     

Identification of promiscuous HPV16‐derived T helper cell epitopes for therapeutic HPV vaccine design

Cervical carcinoma and several other human papillomavirus (HPV)‐induced malignancies are a global public health problem, thus novel treatment modalities are urgently needed. Immunotherapy is an attractive option for treatment of HPV infection and HPV‐mediated premalignant and malignant lesions. However, previous approaches—focusing on the induction of cytotoxic CD8+ T cells (CTLs)—have as yet not yielded clinical successes. Since CD4+ T cells have been shown to be crucial for the induction and maintenance of CTL responses, and more recently to be also important for direct anti‐tumor immunity, human leukocyte antigen (HLA) class II‐restricted epitopes are intensively investigated to improve the efficacy of peptide‐based HPV immunotherapy. We here present an approach to identify promiscuous HPV16‐derived CD4+ T helper epitopes, which are capable of inducing T cell immunity in a large proportion of the population. To this end, we combined HLA class II epitope prediction servers with in vitro immunological evaluation to identify HPV16 E2‐, E5‐, E6‐, and E7‐derived CD4+ T cell epitopes. Candidate selected HPV16‐derived epitopes were found to be restricted by up to nine HLA‐DR molecules. Furthermore, they were found to induce frequent and robust HPV16 peptide‐specific Th1 responses in healthy donors, as monitored by interferon (IFN)‐γ ELISPOT and cytokine secretion assays. Moreover, these selected peptides also induced specific IFN‐γ T cell responses in blood from HPV16+ CIN2/3 and cervical carcinoma patients. We thus conclude that the identified T helper epitopes are valuable candidates for the development of a comprehensive therapeutic HPV vaccine.     

Induction of human papillomavirus oncogene‐specific CD8 T‐cell effector responses in the genital mucosa of vaccinated mice

Cervical cancer, the second leading cause of cancer mortality in women worldwide, results from infection with a subset of human papillomaviruses (HPV), HPV‐16 being the most prevalent type. The available prophylactic vaccines are an effective strategy to prevent this cancer in the long term. However, they only target 70–80% of all cervical cancers and cannot control existing HPV infections and associated lesions. Therapeutic vaccines are thus necessary for women who cannot benefit from prophylactic vaccination. Induction of protective immune responses in the genital mucosa (GM) may be crucial for efficacy of HPV therapeutic vaccines. We report here that mice that received a single subcutaneous (s.c.) vaccination of an adjuvanted long synthetic HPV16 E7_1–98 polypeptide showed induction of 100% tumor protection against s.c. TC‐1 tumors and that tumor regression was mainly provided by CD8 T cells. In vivo cytotoxic assay revealed high E7‐specific cytolytic T lymphocytes activity in spleen and in genital draining lymph nodes (LN), and E7‐specific CD8 T cells could be detected in GM by tetramer staining. More importantly, high‐avidity E7‐specific INF‐γ secreting CD8 T cells were induced not only in blood, spleen and LN but also in GM of vaccinated mice, thus providing evidence that a parenteral vaccination may be sufficient to provide regression of genital tumors. In addition, there was no correlation between the responses measured in blood with those measured in GM, highlighting the necessity and relevance to determine the immune responses in the mucosa where HPV‐tumors reside.     

Identification of NY‐BR‐1‐specific CD4+ T cell epitopes using HLA‐transgenic mice

Breast cancer represents the second most common cancer type worldwide and has remained the leading cause of cancer‐related deaths among women. The differentiation antigen NY‐BR‐1 appears overexpressed in invasive mammary carcinomas compared to healthy breast tissue, thus representing a promising target antigen for T cell based tumor immunotherapy approaches. Since efficient immune attack of tumors depends on the activity of tumor antigen‐specific CD4⁺ effector T cells, NY‐BR‐1 was screened for the presence of HLA‐restricted CD4⁺ T cell epitopes that could be included in immunological treatment approaches. Upon NY‐BR‐1‐specific DNA immunization of HLA‐transgenic mice and functional ex vivo analysis, a panel of NY‐BR‐1‐derived library peptides was determined that specifically stimulated IFNγ secretion among splenocytes of immunized mice. Following in silico analyses, four candidate epitopes were determined which were successfully used for peptide immunization to establish NY‐BR‐1‐specific, HLA‐DRB1*0301– or HLA‐DRB1*0401‐restricted CD4⁺ T cell lines from splenocytes of peptide immunized HLA‐transgenic mice. Notably, all four CD4⁺ T cell lines recognized human HLA‐DR‐matched dendritic cells (DC) pulsed with lysates of NY‐BR‐1 expressing human tumor cells, demonstrating natural processing of these epitopes also within the human system. Finally, CD4⁺ T cells specific for all four CD4⁺ T cell epitopes were detectable among PBMC of breast cancer patients, showing that CD4⁺ T cell responses against the new epitopes are not deleted nor inactivated by self‐tolerance mechanisms. Our results present the first NY‐BR‐1‐specific HLA‐DRB1*0301– and HLA‐DRB1*0401‐restricted T cell epitopes that could be exploited for therapeutic intervention against breast cancer.     

Effectiveness of novel,lower cost molecular human papillomavirus‐based tests for cervical cancer screening in rural china

This study examined the efficacy of the OncoE6? Cervical Test, careHPV? and visual inspection with acetic acid (VIA) in identifying women at risk for cervical cancer and their capability to detect incident cervical precancer and cancer at 1‐year follow‐up. In a population of 7,543 women living in rural China, women provided a self‐collected and two clinician‐collected specimens and underwent VIA. All screen positive women for any of the tests, a ～10% random sample of test‐negative women that underwent colposcopy at baseline, and an additional ～10% random sample of test‐negative women who did not undergo colposcopy at baseline (n = 3,290) were recruited. 2,904 women were rescreened 1 year later using the same tests, colposcopic referral criteria, and procedures. Sensitivities of baseline tests to detect 1‐year cumulative cervical intraepithelial neoplasia Grade 3 or cancer (CIN3+) were 96.5% and 81.6% for careHPV? on clinician‐collected and self‐collected specimens, respectively, and 54.4% for OncoE6? test. The OncoE6? test was very specific (99.1%) and had the greatest positive predictive value (PPV; 47.7%) for CIN3+. Baseline and 1‐year follow‐up cervical specimens testing HPV DNA positive was sensitive (88.0%) but poorly predictive (5.5–6.0%) of incident CIN2+, whereas testing repeat HPV16, 18 and 45 E6 positive identified only 24.0% of incident CIN2+ but had a predictive value of 33.3%. This study highlights the different utility of HPV DNA and E6 tests, the former as a screening and the latter as a diagnostic test, for detection of cervical precancer and cancer.     

NY‐ESO‐1 specific antibody and cellular responses in melanoma patients primed with NY‐ESO‐1 protein in ISCOMATRIX and boosted with recombinant NY‐ESO‐1 fowlpox virus

Vaccination strategies based on repeated injections of NY‐ESO‐1 protein formulated in ISCOMATRIX particles (NY‐ESO‐1 ISCOMATRIX) have shown to elicit combined NY‐ESO‐1 specific antibody and T cell responses. However, it remains unclear whether heterologous prime‐boost strategies based on the combination with NY‐ESO‐1 ISCOMATRIX with different NY‐ESO‐1 boosting reagents could be used to increase NY‐ESO‐1 CD8⁺ or CD4⁺ T cell responses. To address this question, we carried out a randomized clinical trial in 39 high‐risk, resected melanoma patients vaccinated with NY‐ESO‐1 ISCOMATRIX, and then boosted with repeated injections of either recombinant fowlpox virus encoding full length NY‐ESO‐1 (rF‐NY‐ESO‐1) (Arm A) or NY‐ESO‐1 ISCOMATRIX alone (Arm B). We have comprehensively analyzed NY‐ESO‐1 specific T cells and B cells response in all patients before and after vaccination for a total of seven time points per patient. NY‐ESO‐1 ISCOMATRIX alone elicited a strong NY‐ESO‐1 specific CD4⁺ T cell and antibody response, which was maintained by both regiments at similar levels. However, CD8⁺ T cell responses were significantly boosted in 3 out of 18 patients in Arm A after the first rF‐NY‐ESO‐1 injection and such responses were maintained until the end of the trial, while no patients in Arm B showed similar CD8⁺ T cell responses. In addition, our results clearly identified immunodominant regions in the NY‐ESO‐1 protein: NY‐ESO‐1_79–102 and NY‐ESO‐1_115–138 for CD4+ T cells and NY‐ESO‐1_85–108 for CD8+ T cells in a large proportion of vaccinated patients. These regions of NY‐ESO‐1 protein should be considered in future clinical trials as immunodominant epitopes.