Andreas M. Kaufmann

Vaccination trial with HPV16 L1E7 chimeric virus-like particles in women suffering from high grade cervical intraepithelial neoplasia (CIN 2/3).

Persistent infection with human papillomaviruses (HPV) is a prerequisite for the development of cervical cancer. Vaccination with virus‐like particles (VLP) has demonstrated efficacy in prophylaxis but lacks therapeutic potential. HPV16 L1E7 chimeric virus‐like particles (CVLP) consist of a carboxy‐terminally truncated HPV16L1 protein fused to the amino‐terminal part of the HPV16 E7 protein and self‐assemble by recombinant expression of the fusion protein. The CVLP are able to induce L1‐ and E7‐specific cytotoxic T lymphocytes. We have performed a first clinical trial to gain information about the safety and to generate preliminary data on the therapeutic potential of the CVLP in humans. A randomized, double blind, placebo‐controlled clinical trial has been conducted in 39 HPV16 mono‐infected high grade cervical intraepithelial neoplasia (CIN) patients (CIN 2/3). Two doses (75 μg or 250 μg) of CVLP were applied. The duration of the study was 24 weeks with 2 optional visits after another 12 and 24 weeks. The vaccine showed a very good safety profile with only minor adverse events attributable to the immunization. Antibodies with high titers against HPV16 L1 and low titers against HPV16 E7 as well as cellular immune responses against both proteins were induced. Responses were equivalent for both vaccine concentrations. A trend for histological improvement to CIN 1 or normal was seen in 39% of the patients receiving the vaccine and only 25% of the placebo recipients. Fifty‐six percent of the responders were also HPV16 DNA‐negative by the end of the study. Therefore, we demonstrated evidence for safety and a nonsignificant trend for the clinical efficacy of the HPV16 L1E7 CVLP vaccine.

Dendritic cell-based tumor vaccine for cervical cancer II: results of a clinical pilot study in 15 individual patients

PurposeHuman papillomavirus (HPV) type 16 and 18 are the most prevalent genotypes in cervical cancer. The viral oncoproteins E6 and E7 are considered to be tumor-specific targets for immunotherapy. HPV E7 antigen-loaded autologous dendritic cells (DC) were evaluated as cellular tumor vaccine in a case series of cervical cancer patients.MethodsAutologous monocyte-derived DCs were pulsed with recombinant HPV16 E7 or HPV18 E7 oncoprotein and administered to 15 stage IV cervical cancer patients. Safety, toxicity, and induction of serological and cellular immune responses were monitored.ResultsThe vaccine was well-tolerated and no local or systemic side effects or toxicity were recorded. A specific serologic response was seen in 3/11 evaluated patients. Specific cellular immune responses (4/11) were detected with 2/10 positive de novo reactions plus one boosted preexistent response in proliferation assays and 3/11 in IFN-γ ELISpot assays. A transient drop in tumor marker SCC was observed in 5/9 evaluable patients but did not correlate with markers of the immune response. No objective clinical response was observed. Tumor biopsies available from four patients showed severe or complete loss of HLA expression in three of the advanced tumors.ConclusionAutologous dendritic cells pulsed with HPV E7 protein can induce T cell responses in a portion of late stage cervical cancer patients. Boosting of immune responses by adjuvants and vaccination of tumor HLA-positive patients will be mandatory in future trials.

HPV16 L1E7 chimeric virus-like particles induce specific HLA-restricted T cells in humans after in vitro vaccination

Cervical cancer has been shown to be highly associated with human papillomavirus (HPV) infection. The viral oncogenes E6 and E7 are constantly expressed by the tumor cells and are therefore targets for immunotherapy. In the present study we investigated the potential of HPV16 L1E7 chimeric virus‐like particles (CVLP) to activate specific cytotoxic T lymphocytes in human blood donors. CVLP were expressed by recombinant baculovirus and purified. Direct incubation of freshly isolated peripheral blood lymphocytes (PBL) with CVLP resulted in induction of proliferation and growth of T cell lines. To enhance antigen presentation we also loaded dendritic cells with CVLP and used them to activate naive T cells. Growing cell lines were mainly CD3 positive (>95%) with a predominant CD4‐positive and a minor CD8‐positive component. Analysis of Tcell specificity was carried out by an interferon‐γ ELISpot assay. Dendritic cells pseudoinfected with CVLP or pulsed with human leukocyte antigen (HLA)‐A*0201‐restricted peptide E711–20 or with a newly identified HPV16 peptide L1323–331 were used as stimulator cells. T cells responsive to CVLP were found in the cultures with frequencies of 0.5%–0.7%. Frequencies to peptides were around 0.1%. These T cells had cytolytic activity toward autologous B‐lymphoblastic cell lines either pseudoinfected with CVLP or pulsed with HLA‐A*0201‐restricted peptides. They also lysed the HPV16‐ and HLA‐A*0201‐positive cervical cancer cell line CaSki, whereas HLA‐A*0201‐negative SiHa cells were not lysed. We conclude from our data that CVLP show promise for a therapeutic vaccine in patients with HPV16‐positive cervical intraepithelial neoplasia lesions or cervical cancer.

Dendritic cell-based tumor vaccine for cervical cancer I: in vitro stimulation with recombinant protein-pulsed dendritic cells induces specific T cells to hpv16 E7 or HPV18 E7

PurposeHuman papillomavirus (HPV) type 16 and 18 are the most prevalent genotypes in cervical cancers. The viral oncoproteins E6 and E7 are considered to be tumor-specific targets for immunotherapy. HPV E7 antigen-loaded dendritic cells (DC) were evaluated as cellular tumor vaccine.MethodsAutologous monocyte-derived DCs loaded with recombinant HPV16 or HPV18 E7 oncoprotein were used to induce in vitro a specific T cell response. Specificities of activated T cells were determined.ResultsE7-specific T cells could be identified in 18/20 T cell lines from healthy blood donors. CD4+ T cell responses (13/16) were found by proliferation assay. CD8+ CTLs (12/18) were detectable by interferon-gamma (IFN-γ) ELISpot analysis. Seven donors reacted in both assays and only 2/20 T cell lines did not react in any assay. Thus, specific T cells could be activated in >80% of healthy individuals. T cell lines from suitable donors were specific for HLA-A*0201-restricted epitopes. Furthermore, HPV E7 antigen-loaded DC stimulated specific responses in freshly isolated tumor infiltrating lymphocyte (TIL) populations of cervical cancer patients.ConclusionAutologous dendritic cells loaded with HPV E7 protein can induce T cell responses in healthy individuals by in vitro stimulation and evoke responses in TIL from cervical cancer biopsies. Since there are no limitations with respect to specific HLA-haplotypes, these findings may be a basis for the development of a therapeutic protein-based DC tumor vaccine against cervical cancer for HPV16- and HPV18-positive patients.

T cell response to human papillomavirus 16 E7 in mice: Comparison of Cr release assay, intracellular IFN-γ production, ELISPOT and tetramer staining

Successful vaccination against infections by high-risk papillomaviruses aiming at the prevention of cervical cancer most likely requires the induction of neutralizing antibodies and human papillomavirus (HPV)-specific T cells directed against early viral proteins such as E7. Whereas the technology for detection of antibodies is well established, measurement of T cells is more cumbersome and standardization of assays is difficult. By using chromium release assay, ELISPOT, tetramer staining and intracellular IFN-γ assay, we compared the levels of HPV 16 E7-specific T cells obtained after immunization of C57BL/6 mice with different DNA expression vectors. We found that all four assays gave highly comparable results. ELISPOT can be recommended for future studies as it indicates the presence of activated (i.e. IFN-γ-secreting) T cells in a quantitative manner and combines high sensitivity with relatively low T cell demand.

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 IFNγ‐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.

EPIDEMIOLOGIE, ATIOLOGIE UND PRAVENTION DES ZERVIXKARZINOMS

Durch molekulare und epidemiologische Studien wurde bewiesen, daß die humanpathogenen Papillomviren (HPV) Typ 16 und 18 ursächlich an der Entstehung des Zervixkarzinoms beteiligt sind. Diese Aussage gilt auch in bisher noch eingeschränktem Maße für andere sog. High-risk-HPV-Typen wie HPV 31, 33, 35, 39, 45, 51, 52, 56 und 58. Etwa 1/2 Mio. Frauen erkranken jährlich weltweit am Zervixkarzinom und zwischen 1–4% aller jüngeren Frauen leiden an einer Präkanzerose der Cervix uteri. Das Wissen um die virale Genese anogenitaler Neoplasien wird jedoch bisher nicht für die primäre und sekundäre Prävention dieser Erkrankungen eingesetzt. Dies liegt vor allem daran, daß die Infektion mit genitalen HPV-Typen bei jungen Frauen hoch prävalent ist, jedoch nur selten zum Karzinom führt. In der folgenden Übersicht werden daher die wichtigsten vorliegenden epidemiologischen Daten zum Zervixkarzinom und seinen Vorstufen auf ihre Assoziation mit HPV analysiert. Im weiteren werden die molekularbiologischen Daten von HPV für die ano- genitale Karzinogenese dargestellt. Abschließend wird auf die Bedeutung von HPV für die Prävention des Zervixkarzinoms eingegangen.

Preclinical investigation of DNA immunization with a rearranged human papillomavirus type 16 (HPV16) E7 oncogene

Infection with human papillomaviruses (HPV) is the major risk factor for the development of cervical cancer. The HPV E7 oncogene is constitutively expressed in infected cells hence it represents a promising target for immune therapy of HPV-related disease. For safety reasons the use of a transforming gene for DNA vaccination is not feasible. Therefore, we have generated a rearranged (shuffled) HPV16 E7 artificial gene (HPV16 E7SH) specifically dissected at the sites associated with cell transformation. Sequence duplications were added in order to supply all original T cell epitopes. The potential risk of back to wild type recombination was reduced by the use of different codons in the duplicated sequences.

A refined method for lymphocyte isolation from the placenta

OBJECTIVES:u2002 Analysis of placental lymphocytesfrom different compartments is crucial for many questions in reproductive immunology. A separation of lymphocytes after tissue disaggregations leads to a mixture of maternal and fetal lymphocytes, which are difficult to distinguish by simple methods. HLA‐A2 is expressed by 29% (Romanians) – 45% (Spanish) of a Caucasian population and might be a marker for this approach.

Molekulare Diagnostik und Therapie der HPV-assoziierten genitalen Erkrankungen

Zum ThemaDie molekularbiologische Diagnostik von humanen Papillomvirus (HPV)-Infektionen und HPV-assoziierten Neoplasien des unteren Genitaltrakts erfolgt gegenwärtig ausschließlich durch DNA-Nachweisverfahren. Hierbei kommen sowohl amplifizierende als auch nichtamplifizierende Methoden zum Einsatz. Bei den nichtamplifizierenden Verfahren hat sich der „hybrid capture assay“ durchgesetzt, der kommerziell verfügbar ist und mit dem ein Gemisch von 13 High-risk (HR)- oder 5 Low-risk (LR)-HPV-Typen nachgewiesen werden kann. Der Test ist inzwischen semiautomatisiert und hat sich in mehreren klinischen Studien bewährt. Bei den amplifizierenden Verfahren werden ein System mit degenerierten Konsensusprimern und ein weiteres mit nichtdegenerierten Konsensusprimern angewandt, die beide eine spezifische Sequenz aus dem L1 offenen Leseraster von verschiedenen HPV-Typen amplifizieren und den Nachweis von ca. 10 HPV-DNA-Kopien ermöglichen. Auch mit den amplifizierenden Verfahren kann ein breites Spektrum von HR- und LR-HPV-Typen in einem Test mit hoher Spezifität identifiziert werden.In der klinischen Anwendung kann die HPV-Diagnostik im Rahmen der Vorsorge zur Augmentierung der Zytologie, als Triage für die Abklärung fraglicher zytologischer Veränderungen und als Prognosefaktor für die Einstufung des biologischen Potentials von leichtgradigen Präkanzerosen eingesetzt werden. Mehrere Studien konnten zeigen, daß für die Vorsorge und für die Abklärung unklarer zytologischer Befunde die HPV-Diagnostik eine deutliche Verbesserung der bisher üblichen Algorithmen erlaubt.Bei der Therapie von benignen HPV-assoziierten genitalen Erkrankungen ist die Chemodestruktion mittels Podophyllotoxin oder Trichloressigsäure sowie die physikalische Destruktion mittels CO2-Laser, Kryotherapie oder elektrochirurgischer Abtragung das Mittel der Wahl. Die Behandlung mit Interferon zeigte keinen reproduzierbaren positiven Effekt, potentielle Erfolge mit neuen Immunmodulatoren müssen erst verifiziert werden. Krebsvorstufen sollten nach kolposkopischer und histologischer Evaluierung mittels Knipsbiopsie durch chirurgische Exzision mittels Hochfrequenzschlinge oder CO2-Laser behandelt werden. Beide Verfahren zeigen gegenüber der klassischen Messerkonisation eine geringere peri- und postoperative Morbidität. Interferon sollte bei der adjuvanten Behandlung von Präkanzerosen nur beim Rezidiv oder ausgedehnter Erkrankung eingesetzt werden. Die Wertigkeit neuer Immunmodulatoren wie Leukonorm muß erst in prospektiven randomisierten Studien gezeigt werden.Die ideale Prophylaxe und Therapie von HPV stellt die Impfung dar. Ergebnisse im Tiermodell zeigen, daß eine Prophylaxe möglich ist. Erste Ergebnisse der therapeutischen Vakzinierung beim Menschen sind noch wenig aussagekräftig. Für die Zukunft scheinen chimäre virusähnliche Partikel als idealer Impfstoff, da sie sowohl prophylaktisch als auch therapeutisch wirken können.