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The insight that the immune system is involved in tumor resistance is gaining momentum and this has led to the development of immunotherapeutic strategies aiming at enhancement of immune-mediated tumor destruction. Although some of these strategies have moderate clinical benefit, most stand-alone therapies fail to significantly affect progressive disease and survival or do so only in a minority of patients. Research on the mechanisms underlying the generation of immune responses against tumors and the immune evasion by tumors has emphasized that various mechanisms simultaneously prevent effective immunity against cancer including inefficient presentation of tumor antigens by dendritic cells and induction of negative immune regulation by regulatory T-cells (Tregs) and myeloid derived suppressor cells (MDSCs). Thus the design of therapies that simultaneously improve effective tumor immunity and counteract immune evasion by tumors seems most desirable for clinical efficacy. As it is unlikely that a single immunotherapeutic strategy addresses all necessary requirements, combinatorial strategies that act synergistically need to be developed. Here we discuss the current knowledge and prospects of treatment with synthetic peptide vaccines that stimulate tumor-specific T-cell responses combined with adjuvants, immune modulating antibodies, cytokines and chemotherapy. We conclude that combinatorial approaches have the best potency to accomplish the most significant tumor destruction but further research is required to optimize such approaches.

Background Human papilloma virus type 16 (HPV16)-induced gynecological cancers, in particular cervical cancers, are found in many women worldwide. The HPV16 encoded oncoproteins E6 and E7 are tumor-specific targets for the adaptive immune system permitting the development of an HPV16-synthetic long peptide (SLP) vaccine with an excellent treatment profile in animal models. Here, we determined the toxicity, safety, immunogenicity and efficacy of the HPV16 SLP vaccine in patients with advanced or recurrent HPV16-induced gynecological carcinoma. Methods Patients with HPV16-positive advanced or recurrent gynecological carcinoma (n = 20) were subcutaneously vaccinated with an HPV16-SLP vaccine consisting of a mix of 13 HPV16 E6 and HPV16 E7 overlapping long peptides in Montanide ISA-51 adjuvant. The primary endpoints were safety, toxicity and tumor regression as determined by RECIST. In addition, the vaccine-induced T-cell response was assessed by proliferation and associated cytokine production as well as IFNγ-ELISPOT. Results No systemic toxicity beyond CTCAE grade II was observed. In a few patients transient flu-like symptoms were observed. In 9 out of 16 tested patients vaccine-induced HPV16-specific proliferative responses were detected which were associated with the production of IFNγ, TNFα, IL-5 and/or IL-10. ELISPOT analysis revealed a vaccine-induced immune response in 11 of the 13 tested patients. The capacity to respond to the vaccine was positively correlated to the patient’s immune status as reflected by their response to common recall antigens at the start of the trial. Median survival was 12.6 ± 9.1 months. No regression of tumors was observed among the 12 evaluable patients. Nineteen patients died of progressive disease. Conclusions The HPV16-SLP vaccine was well tolerated and induced a broad IFNγ-associated T-cell response in patients with advanced or recurrent HPV16-induced gynecological carcinoma but neither induced tumor regression nor prevented progressive disease. We, therefore, plan to use this vaccine in combination with chemotherapy and immunomodulation.

We previously established safety and immunogenicity of a p53 synthetic long peptides (p53-SLP (R)) vaccine. In the current trial, we investigated whether combination of interferon-alpha (IFN-a) with p53-SLP (R) is both safe and able to improve the induced p53-specific IFN-? response. Eleven colorectal cancer patients successfully treated for metastatic disease were enrolled in this study. Of these, nine patients completed follow-up after two injections with p53-SLP (R) together with IFN-a. Safety and p53-specific immune responses were determined before and after vaccination. Furthermore, cryopreserved PBMCs were compared head-to-head to cryopreserved PBMCs obtained in our previous trial with p53-SLP (R) only. Toxicity of p53-SLP (R) vaccination in combination with IFN-a was limited to Grade 1 or 2, with predominantly small ongoing swellings at the vaccination site. All patients harbored p53-specific T cells after vaccination and most patients showed p53-specific antibodies. Compared to the previous trial, addition of IFN-a significantly improved the frequency of p53-specific T cells in IFN-? ELISPOT. Moreover, in this trial, p53-specific T cells were detectable in blood samples of all patients in a direct ex vivo multiparameter flowcytometric assay, opposed to only 2 of 10 patients vaccinated with p53-SLP (R) only. Finally, patients in this trial displayed a broader p53-specific immunoglobulin-G response, indicating an overall better p53-specific T-helper response. Our study shows that p53-SLP (R) vaccination combined with IFN-a injection is safe and capable of inducing p53-specific immunity. When compared to a similar trial with p53-SLP (R) vaccination alone the combination was found to induce significantly more IFN-? producing p53-specific T cells.

The efficiency of antigen (Ag) processing by dendritic cells (DCs) is vital for the strength of the ensuing T-cell responses. Previously, we and others have shown that in comparison to protein vaccines, vaccination with synthetic long peptides (SLPs) has shown more promising (pre-)clinical results. Here, we studied the unknown mechanisms underlying the observed vaccine efficacy of SLPs. We report an in vitro processing analysis of SLPs for MHC class I and class II presentation by murine DCs and human monocyte-derived DCs. Compared to protein, SLPs were rapidly and much more efficiently processed by DCs, resulting in an increased presentation to CD4⁺ and CD8⁺ T cells. The mechanism of access to MHC class I loading appeared to differ between the two forms of Ag. Whereas whole soluble protein Ag ended up largely in endolysosomes, SLPs were detected very rapidly outside the endolysosomes after internalization by DCs, followed by proteasome- and transporter associated with Ag processing-dependent MHC class I presentation. Compared to the slower processing route taken by whole protein Ags, our results indicate that the efficient internalization of SLPs, accomplished by DCs but not by B or T cells and characterized by a different and faster intracellular routing, leads to enhanced CD8⁺ T-cell activation.

Persistent infection of basal keratinocytes with high-risk human papillomavirus (hrHPV) may cause cancer. Keratinocytes are equipped with different pattern recognition receptors (PRRs) but hrHPV has developed ways to dampen their signals resulting in minimal inflammation and evasion of host immunity for sustained periods of time. To understand the mechanisms underlying hrHPV's capacity to evade immunity, we studied PRR signaling in non, newly, and persistently hrHPV-infected keratinocytes. We found that active infection with hrHPV hampered the relay of signals downstream of the PRRs to the nucleus, thereby affecting the production of type-I interferon and pro-inflammatory cytokines and chemokines. This suppression was shown to depend on hrHPV-induced expression of the cellular protein ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in keratinocytes. UCHL1 accomplished this by inhibiting tumor necrosis factor receptor-associated factor 3 (TRAF3) K63 poly-ubiquitination which lead to lower levels of TRAF3 bound to TANK-binding kinase 1 and a reduced phosphorylation of interferon regulatory factor 3. Furthermore, UCHL1 mediated the degradation of the NF-kappa-B essential modulator with as result the suppression of p65 phosphorylation and canonical NF-κB signaling. We conclude that hrHPV exploits the cellular protein UCHL1 to evade host innate immunity by suppressing PRR-induced keratinocyte-mediated production of interferons, cytokines and chemokines, which normally results in the attraction and activation of an adaptive immune response. This identifies UCHL1 as a negative regulator of PRR-induced immune responses and consequently its virus-increased expression as a strategy for hrHPV to persist., Author Summary A persistent infection with high-risk human papillomavirus (hrHPV) may cause cancer. Whereas keratinocytes – the cells infected by hrHPV – are equipped with different receptors allowing them to recognize invading pathogens and to activate the immune system, hrHPV has developed ways to evade the host's immune response for sustained periods of time. We showed that hrHPV accomplishes this by interfering with the signaling of the pathogen receptors, thereby hampering the production of cytokines that are known to attract and activate the immune system. HrHPV accomplishes this by upregulating the expression of a cellular protein called ubiquitin carboxyl-terminal hydrolase L1 (UCHL1). This protein suppresses the activation of signals downstream of the pathogen receptor leading to reduced transcription factor activation and downstream gene expression, in particular that of type I interferon and pro-inflammatory cytokines. This lowers the attraction of immune cells and thereby the chance of hrHPV-infected cells to be recognized and eliminated and as such enables hrHPV to persist.

A cytotoxic T lymphocyte (CTL) clone generatedin vitrofrom the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant p53 genes, in a L9V/HLA-A2 specific and restricted fashion. Thus, the normal tolerance against endogenously processed p53 protein-derived self-epitopes can be broken by peptide-specificin vitropriming. p53 protein-derived wild-type peptides might thus represent tumor associated target molecules for immunotherapeutical approaches.

Tumor-specific cytotoxic T cells (CTLs) can play an important role against cancer as illustrated by the observation that adoptive transfer of tumor-specific CTLs can mediate potent anti-tumor effects. Although such CTLs can be detected at the tumor site, relatively little is known about the mechanisms by which they enter the tumor. In this study, the role of major histocompatibility complex (MHC) class 1 molecules on vascular endothelium in the tumor in entry of, and tumor eradication by, tumor-specific CTL was investigated. Two H-2Db-restricted CTL clones recognizing peptide VNIRNCCYI on human adenovirus type 5 early region 1-(Ad5E1)-induced tumors were used to test whether CTLs were able to cross the vascular endothelium lacking the restricting MHC molecule. One CTL clone recognizes peptide VNIRNCCYI in the context of both H-2Db and H-2Dbm14 molecules. The other CTL clone recognizes this peptide only in the context of H-2Db. Adoptive transfer of these CTLs leads to eradication of Ad5E 1-induced, H-2Db-expressing tumors in B6(H-2Db+) and Bm14(H-2Db-) nude mice. Our data show that presentation of tumor-derived peptides by MHC molecules on endothelial cells of blood vessels in a tumor do not play a major role in eradication of tumors by adoptively transferred CTL in combination with interleukin-2. Moreover, our data show that successful adoptive CTL immunotherapy is possible across allogeneic barriers, without inducing severe side effects, provided the tumor expresses the MHC class 1-peptide complex recognized by the CTLs.

T cell-mediated immunity plays an important role in the defense against human papillomavirus (HPV) associated cervical cancer as evident from data of the etiology of the virus, several animal models and clinical data. This review addresses the most recent observations in this field which have led to the first clinical trials that are aimed at an immunotherapeutic intervention in this disease via induction of HPV specific T-cell responses.

Antigen-presenting cells contain a specialized late endocytic compartment, MIIC (major histocompatibility complex [MHC] class II-enriched compartment), that harbors newly synthesized MHC class II molecules in transit to the plasma membrane. MIICs have a limiting membrane enclosing characteristic internal membrane vesicles. Both the limiting membrane and the internal vesicles contain MHC class II. In this study on B lymphoblastoid cells, we demonstrate by immunoelectron microscopy that the limiting membrane of MIICs can fuse directly with the plasma membrane, resulting in release from the cells of internal MHC class II-containing vesicles. These secreted vesicles, named exosomes, were isolated from the cell culture media by differential centrifugation followed by flotation on sucrose density gradients. The overall surface protein composition of exosomes differed significantly from that of the plasma membrane. Exosome-bound MHC class II was in a compact, peptide-bound conformation. Metabolically labeled MHC class II was released into the extracellular medium with relatively slow kinetics, 10 +/- 4% in 24 h, indicating that direct fusion of MIICs with the plasma membrane is not the major pathway by which MHC class II reaches the plasma membrane. Exosomes derived from both human and murine B lymphocytes induced antigen-specific MHC class II-restricted T cell responses. These data suggest a role for exosomes in antigen presentation in vivo.

Functional polymorphism in the rat peptide transporter associated with antigen processing (TAP) changes the peptide pool available for binding and presentation by a class I MHC allele, RT1.Aa. The peptide binding motif for RT1.Aa, determined by stabilization with synthetic peptides, included a strong preference for arginine at the peptide C terminus. Analysis of natural peptides bound to RT1.Aa by both pool sequencing and anhydrotrypsin chromatography revealed that TAP polymorphism determined the presence or absence of arginine as the peptide C-terminal residue. This result highlights the in vivo impact of TAP–peptide selectivity, and provides evidence against a high rate of generation of new C termini by protease activity in the endoplasmic reticulum.

Previously, we have shown that immunization with human papillomavirus (HPV) type 16-derived cytotoxic T lymphocyte (CTL) epitope E7 49-57 (RAHYNIVTF) renders C57BL/6 mice insensitive to tumors formed by HPV16-transformed cells. In this study, we provide evidence that E7 49-57 is expressed as a subdominant CTL epitope on HPV16-transformed C57BL/6 cells. Using acid peptide elution, it is shown that HPV16-transformed cells express another CTL epitope, besides E7 49-57, which appears to be dominant. We demonstrate that a CTL line raised against the subdominant CTL epitope, offered as synthetic peptide E7 49-57, eradicates established HPV16-induced tumors in mice. Our data show that synthetic peptide-induced CTL can be applied successfully in vivo against (virus-induced) tumor, and emphasize that subdominant CTL epitopes are useful targets for immunotherapy. Furthermore, it is illustrated for the first time that HPV16-specific CTL interfere directly with HPV16-induced tumors.

Cancer antigens of choice include mutant DNA sequences encoding neo-antigens, selected cancer testis antigens, and differentiation antigens, in addition to viral antigens. Neo-antigens and viral antigens are the most attractive, because the T cell repertoire against these antigens has not been blunted by central thymic tolerance. Optimal vaccine platforms include DNA and RNA vaccines as well as synthetic long peptides and selected prime-boost protocols involving recombinant viruses encoding cancer antigens. The most effective cancer vaccines deliver concentrated antigen to both HLA class I and II molecules of DCs, promoting both CD4 and CD8 T cell responses. Suboptimal antigen selection, suboptimal vaccine design, and an immunosuppressive cancer microenvironment are the root causes of the lack of clinical efficacy of cancer vaccines in many clinical trials. Drugs or physical treatments can mitigate the immunosuppressive cancer microenvironment and include chemotherapeutics, radiation, inhibitors of T cell checkpoints, indoleamine 2,3-dioxygenase (IDO) inhibitors, agonists of selected TNF receptor family members, and inhibitors of undesirable cytokines. The specificity of therapeutic vaccination combined with such immunomodulation offers an attractive avenue for the development of future cancer therapies. Clinical benefit of certain therapeutic cancer vaccines has been established, usually by noting prolonged progression free or overall survival. Reviews of the properties and prospects of cancer vaccines have been listed below (1-3). The addition of one or more immunomodulation strategies may further enhance the potential of therapeutic cancer vaccines, including of those that have been only marginally active due to the immunosuppressive cancer microenvironment. We have established clinical benefit of a cancer vaccine as monotherapy in patients with premalignant disease induced by high risk human papilloma virus. Regression of lesions was associated with a better vaccine-induced immune response against the target antigens of the vaccine. However in patients with cervical cancer the vaccine monotherapy failed to install clinically effective immunity. Recently we showed in a pilot trial that vaccine-induced immunity could be markedly enhanced by combination with standard of care chemotherapy for late stage recurrent or metastatic cervical cancer. Increased levels of myeloid-derived suppressor cells were shown to be depleted by the carboplatin and paclitaxel chemotherapy to the low levels of healthy donors, while T cell numbers in PBMC remained intact. Timed vaccination with a single dose of vaccine at the MDSC nadir during chemotherapy led to a robust vaccine-induced T cell response (4). In a subsequent chemo-immunotherapy trial with three vaccine doses, spaced by three weeks, and in cohorts of patients at four different vaccine doses, the initiation and maintenance of robust tumor-specific T cell responses was confirmed and a broad dose response relationship of tumor-specific T cell responses was noted. Preliminary data on clinical outcomes relative to HPV-specific immune responses will be discussed. In summary, therapeutic benefit of cancer vaccines can be maximized in patients with established cancers by improving vaccine design and by using vaccines in combination with standard chemotherapies. In addition cancer vaccines can establish and/or maintain tumor-specific T cells in combination with checkpoint blockade or other immunomodulatory therapies. Finally, cancer vaccines can also help to sustain and expand adoptively transferred tumor-specific T cells. References 1. Melero I, Gaudernack G, Gerritsen W, Huber C, Parmiani G, Scholl S, Thatcher N, Wagstaff J, Zielinski C, Faulkner I, Mellstedt H, Therapeutic vaccines for cancer: an overview of clinical trials. Nat Rev Clin Oncol. 2014;11(9):509-524. 2. Melief CJ,.van Hall T, Arens R, Ossendorp F, van der Burg SH. Therapeutic cancer vaccines. J Clin Invest. 2015 125(9):3401-3412. 3. van der Burg SH, Arens R, Ossendorp F, van Hall T, Melief CJ. Vaccines for established cancer: overcoming the challenges posed by immune evasion. Nat Rev.Cancer. 2016 Apr;16(4):219-33. 4. Welters MJ, van der Sluis TC, van Meir H, Loof NM, van Ham VJ, van Duikeren S, Santegoets SJ, Arens R, de Kam ML, Cohen AF, van Poelgeest MI, Kenter GG, Kroep JR, Burggraaf J, Melief CJ, van der Burg SH. Vaccination during myeloid cell depletion by cancer chemotherapy fosters robust T cell responses. Sci Transl Med. 2016 Apr 8(334):334ra52. Citation Format: Cornelis J. M. Melief. Selection of cancer antigens and conditions for success of immunotherapy of cancer involving vaccination [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA02.

Monoclonal antibody (mAb) blockade of immune checkpoints such as PD-1 and CTLA-4 can stimulate therapeutic, T cell-dependent anti-tumor activity in mice and humans. We asked whether exome sequencing and epitope prediction algorithms could identify antigens in progressively growing tumors that render them susceptible to anti-PD-1- or anti-CTLA-4-induced immune elimination. Expressed mutations in progressively growing, T3 methylcholanthrene (MCA)-sarcomas were identified by exome sequencing and prioritized based primarily on potential MHC class I binding affinity and predicted peptide processing. The two best candidate tumor-specific mutant antigens (TSMA) were identified from mutations in Laminin α subunit 4 (Lama4) and a glucosyltransferase called Alg8. When tested in vitro, mutant Lama4 (mLama4) and mutant Alg8 (mAlg8) peptides were the only top predicted TSMA that stimulated T3-specific T cells. The relevance of these findings was validated by four in vivo studies: (i) mLama4 and mAlg8 peptides bound to H-2Kb were biochemically detected on the surface of T3 tumors, (ii) as shown by tetramer staining, CD8+ tumor infiltrating lymphocytes (TIL) expressing TCRs for mLama4 and mAlg8 accumulated over time in T3 tumors in checkpoint blockade treated, tumor-bearing mice, reaching maximum levels just prior to tumor rejection, (iii) vaccination of naïve wild type mice with mutant but not WT forms of Lama4 or Alg8 induced strong CD8+ T cell responses and (iv) prophylactic or therapeutic vaccination with mLama4 plus mAlg8 synthetic long peptides (SLP) resulted in rejection of T3 cells. The therapeutic protection provided by the SLP vaccine was equal to that afforded by checkpoint blockade therapy. Since we did not see evidence of T cell responses to any other TSMA, we then explored whether the immunodominant antigens masked responses to other TSMA. Using CRISPR/Cas9, we introduced point mutations to revert mLama4 and mAlg8 in T3 back to their nonimmunogenic wild type counterparts. T3 tumor cells with either mLama4 or mAlg8 reverted to wild type were still susceptible to anti-PD-1 or anti-CTLA-4 immune-mediated control with skewing of the T cell response in the TIL towards the remaining TSMA. Additionally, we have obtained T3 tumor cells with both TSMA reverted to wild type and are currently testing whether these “fixed” T3 tumor cells are still subject to anti-PD-1 or anti-CTLA-4 and whether SLP vaccines comprised of subdominant TSMA still present in fixed T3 tumor cells can protect mice against tumor growth. This system will allow us to address the many questions surrounding poorly understood phenomenon of immunodominance and how this may influence patient-specific personalized cancer immunotherapy directed against TSMA. Citation Format: Matthew M. Gubin, Jeffrey P. Ward, Takuro Noguchi, Xiuli Zhang, Cora Arthur, Willem-Jan Krebber, Gwenn E. Mulder, Cornelis J.M. Melief, William E. Gillanders, Maxim Artyomov, Elaine R. Mardis, Robert D. Schreiber. Tumor-specific mutant antigens in cancer immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A001.

Previously we have demonstrated that two doses of a cytotoxic T lymphocyte (CTL) epitope-based peptide vaccine of human papillomavirus type 16 (HPV 16) E7 aa 49-57 elicit protection against outgrowth of HPV 16-transformed tumor cells (C3 cells) in B6 mice. Incomplete Freund's adjuvant (IFA), as a carrier, was used to induce this response. To avoid side effects caused by the use of external adjuvants, we have now investigated the effectiveness of highly purified spleen dendritic cells (DC) that efficiently induce primary peptide-specific CTL responses in vitro, as physiological carriers for the HPV 16 E7(49-57) peptide-based vaccine. This is the first report demonstrating that mice immunized once i.v. with syngeneic spleen DCs pulsed with the HPV 16 E7(49-57) peptide in vitro were protected against the outgrowth of C3 tumor cells. In comparison, a single injection of the HPV 16 E7(49-57) peptide in IFA s.c. also resulted in effective induction of tumor-specific immunity in vivo. In both immunization protocols, protective tumor-specific immunity was mediated by CTL that recognized HPV 16 E7(49-57) peptide-pulsed target cells, as well as C3 cells in vitro. Peptide affinity of the CTL induced by both protocols was similar. Thus under the conditions tested, a single injection of spleen DCs pulsed with a CTL epitope-based peptide in vitro elicited tumor-antigen-specific CTL in vivo, which protected mice against a subsequent tumor inoculation. This result indicates that spleen DCs pulsed with a CTL epitope can effectively serve as a tumor-specific vaccine.

Extensive sets of in total about 2000 synthetic peptides were investigated for their binding affinities to HLA-A ∗ 0201. Comparisons of the amino acid compositions of binding to nonbinding sets of peptides provided new information concerning the rules for 9-, 10-, and 11-mer peptide binding at the amino acid level. Preferred primary anchors were shown to depend on peptide length, longer peptides being more demanding in this respect. A clear preference exists for certain amino acids at several nonanchor positions. In addition, the presence of particular amino acids at those positions almost completely precludes peptide binding. We found no evidence for preferred anchor pairs. From these results new and detailed HLA-A ∗ 0201 peptide-binding motifs for 9-, 10-, and 11-mer peptide binding were deduced. The motifs are in accordance with earlier reports but include new findings, including C as a C-terminal anchor, the importance of D at position 4 for binding, and the deleterious effect of R at position 5 (in 9-mers). The motifs are presented in such a way that they can be used to predict peptide binding to HLA-A ∗ 0201 by computer analysis (see accompanying paper [56]). Human Immunology 43, 1–12 (1995)

Vaccination with peptides recognized by antigen-specific CTLs can prevent lethal virus infections and tumor growth. In order to avoid the synthesis and testing of the numerous overlapping peptide of long AA sequences of proteins of interest, we developed a computer program which utilizes the rules, "motifs" which govern how peptides bind to HLA class I molecules, to derive a predicted binding score for each overlapping peptide. Correlations between the predicted and actual binding results to HLA-A*0201 for 100 peptides selected from six early and two late protein sequences of human papillomavirus type 1a revealed an acceptable level (61%) of concordance. The program is very flexible with regard to the input of protein sequences and motif definitions and is able to handle various motif and peptide lengths.

CD8+ T cells have the potential to attack and eradicate cancer cells. The efficacy of therapeutic vaccines against cancer, however, lacks defined immune correlates of tumor eradication after (therapeutic) vaccination based on features of Ag-specific T cell responses. In this study, we examined CD8+ T cell responses elicited by various peptide and TLR agonist-based vaccine formulations in nontumor settings and show that the formation of CD62L−KLRG1+ effector-memory CD8+ T cells producing the effector cytokines IFN-γ and TNF predicts the degree of therapeutic efficacy of these vaccines against established s.c. tumors. Thus, characteristics of vaccine-induced CD8+ T cell responses instill a predictive determinant for the efficacy of vaccines during tumor therapy.

The aim of this study was to investigate the capacity of an HPV16 E6/E7 synthetic overlapping long-peptide vaccine to stimulate the HPV16-specific T-cell response, to enhance the infiltration of HPV16-specific type 1 T cells into the lesions of patients with HPV16+ high-grade cervical squamous intraepithelial lesion (HSIL) and HPV clearance. This was a placebo-controlled randomized phase II study in patients with HPV16-positive HSIL. HPV16-specific T-cell responses were determined pre- and post-vaccination by ELISPOT, proliferation assay and cytokine assays in PBMC and HSIL-infiltrating lymphocytes, and delayed-type hypersensitivity skin tests. Motivational problems of this patient group to postpone treatment of their premalignant lesions affected the inclusion rates and caused the study to stop prematurely. Of the accrued patients, 4 received a placebo and 5 received 1–2 vaccinations. Side effects mainly were flu-like symptoms and injection site reactions. A strong HPV-specific IFNγ-associated T-cell response was detected by ELISPOT in all vaccinated patients. The outcome of the skin tests correlated well with the ELISPOT analysis. The cytokine profile associated with HPV16-specific proliferation varied from robust type 1 to dominant type 2 responses. No conclusions could be drawn on vaccine-enhanced T-cell infiltration of the lesion, and there was no HPV clearance at the time of LEEP excision. Thus, vaccination of HSIL patients results in increased HPV16-specific T-cell immunity. Further development of this type of treatment relies on the ability to motivate patients and in the reduction in the side effects. Electronic supplementary material The online version of this article (doi:10.1007/s00262-012-1292-7) contains supplementary material, which is available to authorized users.

Background Because of the extreme variability of the HIV-1 genome, a successful vaccine has to effectively recognize diverse infecting HIV-1 strains in the population and must deal with ongoing virus escape in infected individuals. We have selected conserved regions within SIV, corresponding to HIVconsv (Letourneau PLoS ONE 2007) that show little variation across the different isolates and incorporated these in a peptide based vaccine strategy.

Introduction: In view of the poor prognosis of ovarian cancer, new treatment modalities such as immunotherapy are under investigation. We have shown that a p53-SLP vaccine induces p53-specific T-cell responses. Yet, no clinical responses were observed possibly due to immune suppressive activity of regulatory T-cells (Tregs). Objective: Purpose of this phase II single-arm clinical trial was to evaluate whether administration of low dose cyclophosphamide before vaccination improves immunogenicity of the p53-SLP vaccine in recurrent ovarian cancer patients. Methods: Eleven ovarian cancer patients with recurrent elevation of CA-125 were immunized with the p53-SLP vaccine preceded by low-dose cyclophosphamide. Vaccine-induced p53-specific T-cell responses were evaluated by IFN-γ ELISPOT, proliferation assay, flow cytometry and cytokine bead array. Treg activity was measured by Treg suppression assay. Tumor responses were evaluated with CA-125 levels and CT-scans. Results: Vaccine-induced p53-specific T-cells were observed in 90% (9/10) and 87.5% (7/8) of evaluable patients after two and four immunizations, respectively. Cyclophosphamide induced neither quantitative nor qualitative reduction of Tregs after administration of cyclophosphamide. Nonetheless, the number of vaccine-induced p53-specific T-cells was higher in the current study, compared to our previous study (p ≤ 0.012). Stable disease was observed in 2/10 (20.0%) patients and the remainder of patients (80.0%) showed clinical, biochemical and/or radiographic evidence of progressive disease. Conclusion: Combination of cyclophosphamide with p53-SLP induced no reduction of circulating Tregs. However cyclophosphamide enhanced the induction of a strong p53-specific T-cell response. Our results warrant new studies on the use of low-dose cyclophosphamide to potentiate the immunogenicity of anti-tumor vaccines.

Immunotherapy for ovarian cancer is one of the new treatment strategies currently investigated in epithelial ovarian cancer. This review discusses the results of different immunization strategies, identifies possible drawbacks in study design and provides potential solutions for augmentation of clinical efficacy. A potential target for cancer immunotherapy is p53, as approximately 50% of ovarian cancer cells carry p53 mutations. Therefore we review the immunological and clinical responses observed in ovarian cancer patients vaccinated with p53 targeting vaccines in particular. In most studies antigen-specific vaccine-induced immunological responses were observed. Unfortunately, no clinical responses with significant reduction of tumor-burden have been reported. Based on the currently available results we emphasize the necessity of multimodality treatment of ovarian cancer, combining classical cytoreductive surgery, (neo) adjuvant chemotherapy, immunotherapy and/or targeted therapy.

Synthetic vaccines, in particular long synthetic peptides of approximately 25-50 amino acids in length, are attractive for HIV vaccine development and for induction of therapeutic immune responses in patients with (pre-)malignant disorders. In the case of preventive vaccine development against HIV, no major success has been achieved, but the possibilities are by no means exhausted. A long peptide vaccine consisting of 13 overlapping peptides, which together cover the entire length of the two oncogenic proteins E6 and E7 of high-risk human papilloma virus type 16 (HPV16), caused complete regression of all lesions and eradication of virus in 9 out of 20 women with high-grade vulvar intraepithelial neoplasia, a therapy-resistant preneoplastic disorder. The nature and strength of the vaccine-prompted T cell responses were significantly correlated with the clinical response. Synthetic peptide vaccines are attractive, because they allow rational improvement of vaccine design and detailed pharmacokinetic and pharmacodynamic studies not possible with conventional vaccines. Improvements are possible by addition or conjugation of adjuvants, notably TLR ligands, to the synthetic peptides.

A summit on cellular therapy for cancer discussed and presented advances related to the use of adoptive cellular therapy for melanoma and other cancers. The summit revealed that this field is advancing rapidly. Conventional cellular therapies, such as tumor infiltrating lymphocytes (TIL), are becoming more effective and more available. Gene therapy is becoming an important tool in adoptive cell therapy. Lymphocytes are being engineered to express high affinity T cell receptors (TCRs), chimeric antibody-T cell receptors (CARs) and cytokines. T cell subsets with more naïve and stem cell-like characteristics have been shown in pre-clinical models to be more effective than unselected populations and it is now possible to reprogram T cells and to produce T cells with stem cell characteristics. In the future, combinations of adoptive transfer of T cells and specific vaccination against the cognate antigen can be envisaged to further enhance the effectiveness of these therapies.

Synthetic vaccines, in particular long synthetic peptides of approximately 25-50 amino acids in length, are attractive for HIV vaccine development and for induction of therapeutic immune responses in patients with (pre-)malignant disorders. In the case of preventive vaccine development against HIV, no major success has been achieved, but the possibilities are by no means exhausted. A long peptide vaccine consisting of 13 overlapping peptides, which together cover the entire length of the two oncogenic proteins E6 and E7 of high-risk human papilloma virus type 16 (HPV16), caused complete regression of all lesions and eradication of virus in 9 out of 20 women with high-grade vulvar intraepithelial neoplasia, a therapy-resistant preneoplastic disorder. The nature and strength of the vaccine-prompted T cell responses were significantly correlated with the clinical response. Synthetic peptide vaccines are attractive, because they allow rational improvement of vaccine design and detailed pharmacokinetic and pharmacodynamic studies not possible with conventional vaccines. Improvements are possible by addition or conjugation of adjuvants, notably TLR ligands, to the synthetic peptides.

Cell lineage-specific cellular proteins, oncogenes from viral or cellular origin and tumor suppressor genes encode tumor-specific/associated antigens. Such antigens can elicit an major compatibility complex (MHC) class I-restricted cytotoxic T lymphocyte (CTL) response, either naturally in cancer patients or following appropriate immunostimulation (in vitro or in vivo). The reported immune responses in humans to the melanoma-associated MAGE gene products, GP100 and tyrosinase, all self-proteins, support the idea to use wild-type p53 products as targets for T cells. An important step towards this goal is identification of potential p53 CTL epitopes. We identified the wild-type p53 peptides with the highest affinity to the HLA-A∗0201 molecule using two assays: the previously described MHC peptide-binding assay and the peptide competition assay. We obtained CTL against four p53 peptides with a high affinity for the HLA-A∗0201 molecule. These findings are discussed next to a short review concerning the p53 literature.

Despite the presence of intracellular pathogen recognition receptors that allow infected cells to attract the immune system, undifferentiated keratinocytes (KCs) are the main targets for latent infection with high-risk human papilloma viruses (hrHPVs). HPV infections are transient but on average last for more than one year suggesting that HPV has developed means to evade host immunity. To understand how HPV persists, we studied the innate immune response of undifferentiated human KCs harboring episomal copies of HPV16 and 18 by genome-wide expression profiling. Our data showed that the expression of the different virus-sensing receptors was not affected by the presence of HPV. Poly(I: C) stimulation of the viral RNA receptors TLR3, PKR, MDA5 and RIG-I, the latter of which indirectly senses viral DNA through non-self RNA polymerase III transcripts, showed dampening in downstream signalling of these receptors by HPVs. Many of the genes downregulated in HPV-positive KCs involved components of the antigen presenting pathway, the inflammasome, the production of antivirals, pro-inflammatory and chemotactic cytokines, and components downstream of activated pathogen receptors. Notably, gene and/or protein interaction analysis revealed the downregulation of a network of genes that was strongly interconnected by IL-1 beta, a crucial cytokine to activate adaptive immunity. In summary, our comprehensive expression profiling approach revealed that HPV16 and 18 coordinate a broad deregulation of the keratinocyte's inflammatory response, and contributes to the understanding of virus persistence.

A promising strategy to improve the immunogenic potential of DNA vaccines is the formulation of plasmid DNA (pDNA) with cationic liposomes. In this respect, particle size may be of crucial importance. This study aimed at the evaluation of high-pressure extrusion as a method for sizing cationic liposomes after entrapment of pDNA. This is a well-known sizing method for liposomes, but so far, it has not been applied for liposomes that are already loaded with pDNA. Liposomes composed of egg PC, DOTAP, and DOPE with entrapped pDNA were prepared by the dehydration-rehydration method and subjected to various extrusion cycles, comparing different membrane pore sizes and extrusion frequencies. At optimized extrusion conditions, liposome diameter (Zave) and polydispersity index (PDI) were reduced from 560 nm and 0.56 to 150 nm and 0.14 respectively, and 35% of the pDNA was retained. Importantly, gel electrophoresis and transfection experiments with pDNA extracted from these extruded liposomes demonstrated the preservation of the structural and functional integrity of the pDNA. The reduction in size resulted in enhanced transfection of HeLa cells, as detected by functional expression of the fluorescent protein, eGFP. In addition, these liposomes were able to stimulate Toll-like receptor 9, indicating efficient endosomal uptake and release of the included pDNA. In conclusion, high-pressure extrusion is a suitable technique to size cationic liposomes with entrapped pDNA and allows preparation of well-defined nanosized pDNA-liposomes, with preserved pDNA integrity. Their improved transfection efficiency and ability to activate an important pattern-recognition receptor are favorable properties for DNA vaccine delivery vehicles.

The field of cellular therapy of cancer is moving quickly and the issues involved with its advancement are complex and wide ranging. The growing clinical applications and success of adoptive cellular therapy of cancer has been due to the rapid evolution of immunology, cancer biology, gene therapy and stem cell biology and the translation of advances in these fields from the research laboratory to the clinic. The continued development of this field is dependent on the exchange of ideas across these diverse disciplines, the testing of new ideas in the research laboratory and in animal models, the development of new cellular therapies and GMP methods to produce these therapies, and the testing of new adoptive cell therapies in clinical trials. The Summit on Cell Therapy for Cancer to held on November 1 and 2, 2011 at the National Institutes of Health (NIH) campus will include a mix of perspectives, concepts and ideas related to adoptive cellular therapy that are not normally presented together at any single meeting. This novel assembly will generate new ideas and new collaborations and possibly increase the rate of advancement of this field.

The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples. Electronic supplementary material The online version of this article (doi:10.1007/s00262-012-1251-3) contains supplementary material, which is available to authorized users.

Objectives: Administration of synthetic long peptides (SLPs) derived from human papillomavirus to cervical cancer patients resulted in clinical benefit correlated with expansions of tumour-specific T cells. Because vaginal mucosa is an important port of entry for HIV-1, we have explored SLP for HIV-1 vaccination. Using immunogen HIVconsv derived from the conserved regions of HIV-1, we previously showed in rhesus macaques that SLP.HIVconsv delivered as a boost increased the breath of T-cell specificities elicited by single-gene vaccines. Here, we compared and characterized the use of electroporated pSG2.HIVconsv DNA (D) and imiquimod/montanide-adjuvanted SLP.HIVconsv (S) as priming vaccines for boosting with attenuated chimpanzee adenovirus ChAdV63.HIVconsv (C) and modified vaccinia virus Ankara MVA.HIVconsv (M). Design: Prime-boost regimens of DDDCMS, DSSCMS and SSSCMS in rhesus macaques. Methods: Animals' blood was analysed regularly throughout the vaccination for HIV-1-specific T-cell and antibody responses. Results: We found that electroporation spares DNA dose, both SLP.HIVconsv and pSG2.HIVconsv DNA primed weakly HIVconsv-specific T cells, regimen DDDCM induced the highest frequencies of oligofunctional, proliferating CD4 and CD8 T cells, and a subsequent SLP.HIVconsv boost expanded primarily CD4 cells. DSS was the most efficient regimen inducing antibodies binding to regions of trimeric HIV-1 Env, which are highly conserved among the four major global clades, although no unequivocal neutralizing activity was detected. Conclusion: The present results encourage evaluation of the SLP.HIVconsv vaccine modality in human volunteers along the currently trialled pSG2.HIVconsv DNA, ChAdV63.HIVconsv and MVA.HIVconsv vaccines. These results are discussed in the context of the RV144 trial outcome. © 2012 Wolters Kluwer Health Lippincott Williams and Wilkins.

Until recently, therapeutic cancer vaccines only sporadically led to long-term clinical responses. We here report on a novel vaccine modality, characterized by the administration of long (23-45 amino acids) synthetic peptides in incomplete Freund adjuvant (mineral oil-based, Montanide ISA-51), delivered subcutaneously. Such vaccines were first demonstrated to be much more potent in preclinical T-cell response induction and tumor therapy experiments than short major histocompatibility complex class I-binding peptides that have been used extensively in the clinic. A long-peptide vaccine consisting of 13 overlapping peptides, together covering the entire length of the 2 oncogenic proteins E6 and E7 of high-risk human papilloma virus type 16 (HPV16), caused complete regression of all lesions and eradication of virus in 9 of 20 women with high-grade vulvar intraepithelial neoplasia. The nature and strength of the vaccine-induced T-cell response were significantly correlated with the clinical response. This vaccine promises to be of use, not only in patients with premalignant lesions caused by high-risk HPV16, but also in patients with malignant tumors caused by this virus, including HPV16-positive cervical cancer, anal cancer, and head and neck cancer.

Human Papilloma Virus (HPV)-induced (pre-)malignancies offer an excellent case for the rational design of immunotherapeutic strategies against cancer. Transformed cells express tumor-specific antigens of viral origin while the spontaneous antitumor response and the immunological make up of HPV-induced tumors do not differ from other immunogenic epithelial tumors. A first clinically active therapeutic HPV vaccine has been developed and clear links were found between the type and kinetics of vaccine-induced T-cell immunity. Here, we will review the mechanisms determining the success and failure of therapeutic vaccines against HPV-induced tumors, with particular emphasis on the immunological setting in which these vaccines need to work. The recent progress in therapeutic HPV vaccination will guide the development of successful vaccines in other diseases.

Purpose: Immunotherapy against tumors with anti-CD40 agonistic antibodies has been extensively studied in preclinical animal models and recently also in clinical trials. Although promising results have been obtained, antibody (Ab)-related toxicity has been a limiting factor. We reasoned that strict local activation of tumor-specific CD8 T cells through stimulation of CD40 on the dendritic cells (DC) in the tumor area while excluding systemic stimulation might be sufficient for effective tumor eradication and can limit systemic toxicity. Experimental Design: Preclinical in vivo models for immunogenic tumors were used to investigate the potential of delivering a nontoxic dose of agonistic anti-CD40 Ab to the tumor region, including draining lymph node, in a slow-release formulation (montanide). Results: The delivery of anti-CD40 monoclonal Ab, formulated in slow-release Montanide ISA-51, reprograms CTLs by inducing local but not systemic DC activation, resulting in effective tumor-specific CTL responses that eradicate local and distant tumors. Adverse side effects, assayed by organ histology and liver enzymes in the blood, were much lower after local anti-CD40 Ab delivery than systemic administration. The local delivery of anti-CD40 Ab activates only CTLs against antigens presented in the tumor-draining area, because unrelated distant tumors expressing different tumor antigens were not eradicated. Conclusions: These results establish a novel therapeutic principle that local delivery and slow release of agonistic anti-CD40 Ab to the tumor-draining area effectively activates local tumor-specific CD8 T cells to become systemic effectors without causing systemic toxicity or nonspecific CTL activation. These findings have important implications for the use of anti-CD40 therapies in patients. Clin Cancer Res; 17(8); 2270–80. ©2011 AACR.

Persistent viral infections reflect a failure of the host's immune system to control infection, and in many cases, they are associated with the development of malignancies. So far, vaccines designed to boost viral immunity during chronic infection have not been successful. Infections with high-risk human papilloma viruses (e.g. HPV16) are acquired by a large segment of the population and persist in 5-10% of infected individuals, which causes the development of high-grade pre-malignant lesions. Recently we succeeded in causing regression of HPV16-induced disease in similar to 50% of chronically infected patients by a novel therapeutic vaccine. Here, we summarize the parallels in immunity against HPV and other chronic viruses and discuss the general implications of our findings for the immunotherapy of chronic infections.

Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.

A detailed analyses of HPV-specific immunity was performed in a large group of patients with HPV-induced cervical cancer (CxCa) in relation to HLA-types and prognostic factors. Patients were HLA-typed and HPV16/18-specific T-cell immunity was assessed by proliferation assay and cytometric bead array using freshly isolated PBMC and by phenotypic analysis of HPV-specific T cells. The results were analyzed in relation to known disease-related HLA-types (DR7, DR13, DR15/DQ06), invasion-depth and size of tumor, lymph node (LN) status and disease free survival. In total 119 HLA-typed patients with CxCa were analyzed. Patients expressing the HLA-DR13 haplotype were underrepresented as compared to the Dutch population (p = 0.014), whereas HLA-DR7 was overrepresented in patients with HPV16+ CxCa (p = 0.006). In 29 of 94 patients (31%) from whom blood could be tested, a proliferative response to HPV16/18 was detected, which was associated with increased numbers of HPV-specific CD4+CD25+ (activated) T cells (p = 0.03) and HPV-specific CD4+CD25+FoxP3-positive T cells (p = 0.04). The presence of both FoxP3-positive and negative HPV-specific CD4+CD25+ T cells was significantly correlated (p = 0.01). Interestingly, the detection of HPV-specific proliferation was associated with invasion depth (p = 0.020) but not with HLA type, tumor size nor LN status. Moreover, the detection of HPV-specific immunity was associated with an improved disease free survival (p = 0.04) in patients with deeply infiltrating tumors. In conclusion, HPV-specific proliferative T-cell response, comprising higher percentages of HPV-specific CD25+ and CD25+FoxP3-positive CD4+T cells, are more frequently detected in patients with deep infiltrating CxCa tumors and associated with an improved survival.

Purpose: To predict the potential antitumor effect of antigen-specific T cells in melanoma patients, we investigated T-cell effector function in relation to tumor-escape mechanisms. Experimental Design: CD8+ T cells isolated from tumor, adjacent normal skin, and peripheral blood of 17 HLA-A2+ patients with advanced-stage melanoma were analyzed for their antigen specificity and effector function against melanocyte differentiation antigens MART-1, gp100, and tyrosinase by using HLA-A2/peptide tetramers and functional assays. In addition, the presence of tumor-escape mechanisms PD-L1/PD-1 pathway, FoxP3 and loss of HLA or melanocyte differentiation antigens, both required for tumor cell recognition and killing, were studied. Results: Higher percentages of melanocyte antigen-specific CD8+ T cells were found in the melanoma tissues as compared with adjacent normal skin and peripheral blood. Functional analysis revealed 2 important findings: (i) in 5 of 17 patients, we found cytokine production after specific peptide stimulation by tumor-infiltrating lymphocytes (TIL), not by autologous peripheral blood lymphocytes (PBL); (ii) CD8+ T cells from 7 of 17 patients did not produce cytokines after specific stimulation, which corresponded with significant loss of tumor HLA-A2 expression. The presence of other tumor-escape mechanisms did not correlate to T-cell function. Conclusions: Our data show that functional T-cell responses could be missed when only PBL and not TIL are evaluated, emphasizing the importance of TIL analysis for immunomonitoring. Furthermore, loss of tumor HLA-A2 may explain the lack of T-cell functionality. These findings have important implications for selecting melanoma patients who may benefit from immunotherapy. Clin Cancer Res; 17(17); 5736–47. ©2011 AACR.

The immunogenicity of viral oncoproteins has been established beyond doubt. Cytotoxic T lymphocytes directed against viral oncogene products can eradicate large established tumor masses. This stage has not yet been reached for cellular oncogene and tumor suppressor gene products, but T cells have been raised against MHC-binding peptides encoded by both mutant and wild-type alleles of the ras oncogene and the p53 tumor suppressor gene. In addition, T cells specific for joining region peptides of abnormal fusion proteins resulting from chromosome translocation in tumor cells have been generated. Some of these peptides are processed in cells infected with, for example, vaccinia-ras, but direct anti-tumor effects of peptide specific T lymphocytes remain to be demonstrated.

The central role of the p53 tumor suppressor gene product in oncogenesis is gradually being clarified. Point mutations in the p53 tumor suppressor gene are common in most human cancers and are often associated with p53 protein overexpression. Overexpressed wild-type or mutant determinants of the p53 protein thus represent an attractive target for immunotherapy of cancer directed against a structure involved in malignant transformation. An important step towards this goal is identification of epitopes of p53 that can be recognized by human cytotoxic T lymphocytes. We identified peptides of (mutant) p53 capable of binding to HLA-A2.1 in an in vitro assay. These HLA-A2.1-binding peptides were utilized for in vitro induction of primary cytotoxic T lymphocyte responses using a human processing-defective cell line (174CEM.T2) as antigen-presenting cell. These cells display "empty" HLA class I surface molecules, that can efficiently be loaded with a single peptide. We obtained CD8+ cytotoxic T lymphocyte clones capable of specifically lysing target cells loaded with wild-type or tumor-specific mutant p53 peptides. This strategy allows the in vitro initiation of human cytotoxic T lymphocyte responses against target molecules of choice.

Sendai virus nuclear protein peptides of different lengths were titrated in peptide vaccination experiments. We observed that peptide length was not important in inducing cytotoxic T lymphocyte-mediated protective immunity in vivo against a challenge with a lethal dose of virus. These results suggest that long peptides are trimmed in vivo to peptides that fit into the groove of major histocompatibility complex class I molecules. In addition several adjuvants were screened for their effectiveness in peptide vaccination protocols. Incomplete Freund's adjuvant and Titermax≡4 turned out to be useful, whereas alum was much less effective

Antibody-dependent cellular cytotoxicity (ADCC) is regarded as an important mechanism by which monoclonal antibodies (mAb) can exert an antitumour effect in vivo. It may be possible, therefore, to enhance the therapeutic efficacy of mAb by cytokines that are able to enhance the ADCC of human CD3-, CD56+, CD16+ natural killer (NK) cells. We investigated in vitro the effects of recombinant interferon alpha (rIFN alpha) and recombinant interleukin 2 (rIL-2), alone or in combination, on the ADCC of human peripheral blood NK cells. Both cytokines enhanced the ADCC of the human effector cells. rIFN alpha induced a maximally increased ADCC after an exposure of human effector cells to 20 IU/ml for 15-30 min, while rIL-2 induced optimal ADCC after incubation of the cells for 2 days in 20-50 U/ml. We now show that activation of the NK cells with a combination of rIL-2 and rIFN alpha induced significantly higher levels of ADCC than either cytokine alone. The highest ADCC was induced if the cells were first exposed to rIL-2 before rIFN alpha was added to the culture. Culture of NK cells in medium or rIL-2 decreased the expression of Fc gamma RIII (CD16), indicating that intensity of CD16 expression and level of ADCC are not directly correlated, although blocking experiments with a mAb directed against CD16 showed that this Fc gamma R was essential for ADCC of the human effector cells.

We have developed an in vitro immunization system for the production of B-cell lines that secrete HLA-specific human mAbs. For this purpose, peripheral blood lymphocytes of parous women were stimulated with pools of allogeneic lymphocytes. Preferential outgrowth of B-lymphocytes was effected by inclusion of rIL-2 and a B-cell specific nucleoside analogue. Stimulated B cells were immortalized by EBV transformation, and specific antibody-producing transformants were fused to heteromyeloma or mouse myeloma cell lines, yielding stable hybridomas. This approach has led to the successful development of two human heterohybridomas producing HLA-specific mAbs reactive by complement-mediated cytotoxicity. The specificities of these human mAbs, reactive with HLA-B12(44 + 45) and HLA-B60, respectively, are fully concordant with those of HLA-typing sera.

Cytotoxic T lymphocyte (CTL) peptide epitopes can be used for immunization of mice against lethal virus infection. To study whether this approach can be successful against virus-induced tumors we generated a B6 (H-2b) tumorigenic cell line transformed by human papillomavirus (HPV). This virus is detected in over 90% of all human cervical cancers. To identify vaccine candidates, we generated a set of 240 overlapping peptides derived from the HPV type 16 (HPV16) oncogenes E6 and E7. These peptides were tested for their ability to bind H-2Kb and H-2Db MHC class I molecules. Binding peptides were compared with the presently known peptide-binding motifs for H-2Kb and H-2Db and the predictive value of these motifs is shortly discussed. The high-affinity H-2Db-binding peptide and putative CTL epitope E7 49-57 (RAHYNIVTF) was used in vaccination studies against HPV 16-transformed tumor cells. Immunization with peptide E7 49-57 rendered mice insensitive to a subsequent challenge with HPV 16-transformed tumor cells in vivo, and induced a CTL response which lysed the tumor cells in vitro.

Adoptive T-cell transfer (ACT) is successfully applied as a cancer treatment that is based on the activation and effector functions of tumor-specific T cells. Here, we present results from a mouse model in which ACT is combined with a long peptide–based vaccine comprising gp100 T-cell epitopes. Transferred CD8+ T cells expanded up to 1,000-fold after peptide vaccination, leading to a 3-fold increase in white blood cell count and a very high frequency in the generation of antigen-specific memory T cells, the generation of which tended to correlate with effective antitumor responses. An enormous pool of effector T cells spread widely to different tissues, including the skin and the immune-privileged eye, where they mediate tumor eradication. Importantly, these striking T-cell dynamics occurred in immunocompetent mice without prior hematologic conditioning. Continued activation of the specific T-cell pool by vaccination led to strong T-cell–mediated cytokine storm and lethality due to multi-organ failure. However, this immunopathology could be prevented by controlling the rapid biodistribution of the peptide or by using a weakly agonistic peptide. Together, these results identify a peptide vaccination strategy that can potently accentuate effective ACT in non-lymphodepleted hosts. Cancer Res; 70(21); 8339–46. ©2010 AACR.

Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs), RNA expression arrays, HIV gag expression and crosspresentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferoninduced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activationof pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIVspecific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines.

Priming of naive CD8+ T cells by pathogens or vaccines generally involves their interaction with Ag-loaded dendritic cells (DCs) in the context of an inflamed lymph node. Lymph node activation fosters DC and T cell encounters and subsequently provides newly primed T cells with nurturing conditions. We dissected these two aspects by infusing in vitro primed CD8+ T cells into naive recipient mice harboring a single activated lymph node and comparing the fate of these T cells with those infused into control recipients. Brief (20 h) in vitro priming empowered the T cells to expand in vivo without further Ag stimulation. This primary response was not affected by the presence or absence of a nonspecifically activated lymph node. In contrast, in vivo antigenic challenge after contraction of the primary response resulted in significantly stronger secondary T cell responses in mice harboring activated lymph nodes, demonstrating that the availability of an activated lymph node supported the generation of T cell memory in an Ag-unrelated manner. The presence of an activated lymph node during the expansion and contraction phase of the primary response did not endow T cells with an instructional program for increased survival or secondary expansion, but primarily served to conserve increased numbers of T cells.

One half of a group of 20 patients with human papillomavirus type 16 (HPV16)-induced vulvar intraepithelial neoplasia grade 3 displayed a complete regression (CR) after therapeutic vaccination with HPV16 E6/E7 synthetic long peptides. Patients with relatively larger lesions generally did not display a CR. To investigate immune correlates of treatment failure, patients were grouped according to median lesion size at study entry, and HPV16-specific immunity was analyzed at different time points by complementary immunological assays. The group of patients with smaller lesions displayed stronger and broader vaccine-prompted HPV16-specific proliferative responses with higher IFNγ (P= 0.0003) and IL-5 (P< 0.0001) levels than patients with large lesions. Characteristically, this response was accompanied by a distinct peak in cytokine levels after the first vaccination. In contrast, the patient group with larger lesions mounted higher frequencies of HPV16-specific CD4+CD25+Foxp3+T cells (P= 0.005) and displayed a lower HPV16-specific IFNγ/IL-10 ratio after vaccination (P< 0.01). No disparity in T memory immunity to control antigens was found, indicating that the differences in HPV-specific immunity did not reflect general immune failure. We observed a strong correlation between a defined set of vaccine-prompted specific immune responses and the clinical efficacy of therapeutic vaccination. Notably, a high ratio of HPV16-specific vaccine-prompted effector T cells to HPV16-specific CD4+CD25+Foxp3+T cells was predictive of clinical success. Foxp3+T cells have been associated previously with impaired immunity in malignancies. Here we demonstrate that the vaccine-prompted level of this population is associated with early treatment failure.

Background: Presently melanoma still lacks adequate treatment options for metastatic disease. While melanoma is exceptionally challenging to standard regimens, it is suited for treatment with immunotherapy based on its immunogenicity. Since treatment-related skin depigmentation is considered a favourable prognostic sign during melanoma intervention, we here aimed at the reverse approach of directly inducing vitiligo as a shortcut to effective anti-melanoma immunity.Methodology and Principal Findings: We developed an effective and simple to use form of immunotherapy by combining the topical skin-bleaching agent monobenzone with immune-stimulatory imiquimod cream and cytosine-guanine oligodeoxynucleotides (CpG) injections (MIC therapy). This powerful new approach promptly induced a melanoma antigen-specific immune response, which abolished subcutaneous B16. F10 melanoma growth in up to 85% of C57BL/6 mice. Importantly, this regimen induced over 100 days of tumor-free survival in up to 60% of the mice, and forcefully suppressed tumor growth upon re-challenge either 65- or 165 days after MIC treatment cessation.Conclusions: MIC therapy is effective in eradicating melanoma, by vigilantly incorporating NK-, B-and T cells in its therapeutic effect. Based on these results, the MIC regimen presents a high-yield, low-cost and simple therapy, readily applicable in the clinic