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During the COVID-19 pandemic, the early emergence of viral variants repeatedly undermined the effects of vaccination. Our aim here is to explore strategies for improving spike vaccine gene antigenicity by merging mutations from different variants of concern (VOCs) in a single vaccine gene. To this end, newly developed recombinant vaccine genes were designed, cloned into adenoviral vectors, and applied to C57BL/6 mice; then, serum-neutralizing antibodies against the wildtype SARS-CoV-2 strains were determined in neutralization assays. The merger of mutations from different variants of concern (alpha, beta, gamma, and delta) in a single recombinant spike-based vaccine gene provided a substantial improvement in neutralizing immunity to all variants of concern, including the omicron strains. To date, only unmodified spike genes of the original SARS-CoV-2 Wuhan strain (B.1) or dominant variants (BA.1, BA.5, and XBB.1.5) have been used as vaccine genes. The employment of unmodified vaccine genes is afflicted by limited cross-protection among variant strains. In contrast, recombinant vaccine genes that combine mutations from different strains in a single gene hold the potential to broaden and improve immune protection and might help to reduce the need for frequent vaccine adaptations in the future.

Vaccines play a critical role in combating the COVID-19 pandemic. Future control of the pandemic requires improved vaccines with high efficacy against newly emerging SARS-CoV-2 variants and the ability to reduce virus transmission. Here we compare immune responses and preclinical efficacy of the mRNA vaccine BNT162b2, the adenovirus-vectored spike vaccine Ad2-spike and the live-attenuated virus vaccine candidate sCPD9 in Syrian hamsters, using both homogeneous and heterologous vaccination regimens. Comparative vaccine efficacy was assessed by employing readouts from virus titrations to single-cell RNA sequencing. Our results show that sCPD9 vaccination elicited the most robust immunity, including rapid viral clearance, reduced tissue damage, fast differentiation of pre-plasmablasts, strong systemic and mucosal humoral responses, and rapid recall of memory T cells from lung tissue after challenge with heterologous SARS-CoV-2. Overall, our results demonstrate that live-attenuated vaccines offer advantages over currently available COVID-19 vaccines., (© 2023. The Author(s).)

With the exception of inactivated vaccines, all SARS-CoV-2 vaccines currently used for clinical application focus on the spike envelope glycoprotein as a virus-specific antigen. Compared to other SARS-CoV-2 genes, mutations in the spike protein gene are more rapidly selected and spread within the population, which carries the risk of impairing the efficacy of spike-based vaccines. It is unclear to what extent the loss of neutralizing antibody epitopes can be compensated by cellular immune responses, and whether the use of other SARS-CoV-2 antigens might cause a more diverse immune response and better long-term protection, particularly in light of the continued evolution towards new SARS-CoV-2 variants. To address this question, we explored immunogenicity and protective effects of adenoviral vectors encoding either the full-length spike protein (S), the nucleocapsid protein (N), the receptor binding domain (RBD) or a hybrid construct of RBD and the membrane protein (M) in a highly susceptible COVID-19 hamster model. All adenoviral vaccines provided life-saving protection against SARS-CoV-2-infection. The most efficient protection was achieved after exposure to full-length spike. However, the nucleocapsid protein, which triggered a robust T-cell response but did not facilitate the formation of neutralizing antibodies, controlled early virus replication efficiently and prevented severe pneumonia. Although the full-length spike protein is an excellent target for vaccines, it does not appear to be the only option for future vaccine design.

Purpose: The cell surface glycoprotein Mesothelin is overexpressed in ovarian, fallopian tube, endometrial, cervical and primary peritoneal cancer and, therefore, might become a particular interesting tumor target in gynecologic oncology. However, even in malignant tumors of the same entity the level of Mesothelin expression varies between individuals, hence it can be expected that the response to Mesothelin-targeting therapies will be variable as well. In this study we explored the therapeutic potency of a novel anti-Mesothelin antibody-drug conjugate (Anetumab ravtansine) as a function of Mesothelin expression in the targeted tumor cells., Methods: Anti-tumor activity studies were performed in human uterine xenograft tumor models that express Mesothelin at high, moderate or low levels. The antibody-drug conjugate (ADC) was applied in varying doses ranging from 2 to 15 mg/kg at variable intervals in tumor bearing SCID/beige mice and the impact on tumor growth was monitored., Results: The therapeutic response to the anti-Mesothelin ADC correlated closely with the level of Mesothelin expression in tumor cells. Within the applied dose levels complete tumor regression was achieved only in tumors which expressed Mesothelin at particularly high levels (Hela cell tumors). The application of high anti-Mesothelin ADC doses less frequently was more efficious than giving lower doses at a higher frequency., Conclusion: The studies confirm the great therapeutic potential of Anetumab ravtansine. However, a favorable treatment outcome requires strong Mesothelin expression in tumor cells. Future clinical trials may benefit from a more rigorous selection of appropriate patients based on the level of Mesothelin expression in their tumor tissue. If, in addition, it is possible to better control side effects by introducing protective measures and by doing so to increase the maximum tolerated dose, Anetumab ravtansine has the potency to become a valuable therapeutic tool, especially in the field of gynecological oncology.

Purpose: The cell surface glycoprotein Mesothelin is overexpressed in several tumor entities and novel immune-based therapies are currently under the early clinical evaluation for the treatment of malignant pleura mesothelioma, ovarian cancer, and pancreatic cancer. Cervical cancer has not been recognized as a suitable target for Mesothelin-directed immune therapies so far., Methods: To exploit a possible role of Mesothelin in cervical cancer treatment, we analysed Mesothelin expression in 79 cervical carcinomas and aligned expressions patterns with tumor growth parameters. A novel anti-Mesothelin drug conjugate (Anetumab Ravtansine) was applied for dose-efficiency studies in a Mesothelin positive tumor model for cervical cancer in Scid mice., Results: In more than three-quarters (77%) of cervical adenocarcinomas, Mesothelin was expressed to high levels. Among squamous cell carcinomas of the cervix uteri expression levels were lower and expression patterns were less intense, but still ranged between 50-60% (57%). A significant correlation between Mesothelin expression levels and tumor grade, metastatic behaviour, and lymph- or hemangiosis was not found. The novel anti-Mesothelin-drug conjugate (Anetumab Ravtansine) showed a substantial dose-dependent therapeutic efficiency in a xenotransplant model for cervical cancer in SCID mice (hela cell tumors). Applying the ADC at a dose of 10 mg/kg twice weekly induced complete tumor regression in 88% of animals within 6 weeks., Conclusions: Mesothelin should be taken into account as a target in cervical cancer therapy and histological determination of Mesothelin expression should be considered in routine diagnostics of cervical carcinomas.

Purpose: The identification of novel cell lines which combine the most important properties of mucosal membranes in terms of drug absorption, transmembrane transport and mucus secretion can help to establish improved and meaningful test systems for pharmacological and infectiological studies., Methods: We have established a novel mucus secreting tumor cell line (Cx-03) derived from a female patient who underwent radical hysterectomy after diagnosis of a large malignant carcino sarcoma (Muellerian mixed tumor). Via xenotransplantation in SCID beige mice, recultivation and subcloning a stable cell line was established from primary tumor cells., Results: Human origin and novelty of the cell line was determined by karyotype analysis and STR fingerprint. During growth cells produce considerable amounts of a PAS positive viscoelastic mucus. Immunostaining revealed expression of mucins and the mucin modifier CLCA1. We demonstrate in initial electrophysiological experiments that confluent, polarized monolayers of Cx-03 are formed (on PCF-filter supports) that exhibit stable electrical resistance (> 600 Ω cm 2 ). Confluent Cx-03 monolayers express barrier-forming tight junction proteins claudin-1 and -4 which co-localize with zonula occludens protein-1 (ZO-1) at cell-cell contacts., Conclusions: Mucus secretion is a rare property among mammalian cell lines. In combination with its ability to form polarized monolayers Cx-03 might contribute as a novel cell based model for drug absorption, transport and barrier studies.

Objective: To evaluate the association of three pathognomonic criteria, inner border, ridge sign, and rag sign with high-grade cervical intraepithelial neoplasia (CIN) using video exoscopy., Methods: Retrospective evaluation of video recordings of 335 patients, referred for diagnostic colposcopy, who underwent cervical biopsies, and, if indicated loop excisions, was performed. The most severe histologic diagnosis was recorded. Sensitivity, specificity, positive, negative predictive value, and likelihood ratios for high-grade CIN were calculated., Results: In 285 patients (85%), a single colposcopy directed biopsy was taken; 50 patients (15%) underwent two biopsies. One hundred sixty-two patients (48%) underwent subsequent magnification-guided loop excision. Sensitivity, specificity, positive predictive value, and negative predictive value of the inner border to detect high-grade CIN were 20%, 99%, 97.9%, and 34.8%, respectively. The positive likelihood ratio (LR+) was 20.3 and the negative likelihood ratio (LR-) was 0.81. Sensitivity, specificity, positive predictive value, and negative predictive value of the ridge sign to detect high-grade CIN were 52.5%, 96.4%, 96.8%, and 46.6%, respectively. The LR+ ratio was 13.2 and the LR- ratio was 0.49. Sensitivity, specificity, positive predictive value, and negative predictive value of the rag sign to detect high-grade CIN were 38.4%, 96%, 95.7%, and 40.2%, respectively. The LR+ ratio was 9.7 and the LR- ratio was 0.6. Only the ridge sign showed a correlation with young age. Presence of any two signs significantly increased the LR of the presence of high-grade CIN., Conclusion: The inner border, ridge sign, and the newly defined rag sign are objective, effective colposcopic signs and are significantly associated with high-grade CIN.

Objective: : Our aim was to present our initial clinical experience using a novel exoscopically based colposcopy system (VITOM) for the evaluation of cervical, vulvar, and vaginal diseases., Materials and Methods: : Women referred to the Charite Cervix Center, Charite University, Berlin, Germany, were included. Patients with abnormal Pap smear results, vulvar lesions, or a biopsy report of neoplasia of the lower genital tract were included into the study. The VITOM was used for colposcopic evaluation and directed biopsies. Colposcopic findings were reported according to the criteria of the Committee on Nomenclature of the International Federation of Cervical Pathology and Colposcopy. Histologic diagnosis was described as normal, low-grade lesion, high-grade lesion (including cervical intraepithelial neoplasia 2,3, vulvar intraepithelial neoplasia 2,3, vaginal intraepithelial neoplasia 2,3), or cancer., Results: : We recruited 76 patients (54 with cervical, 4 with vaginal, and 18 with vulvar disease) to the prospective study. Four patients were pregnant. Of patients with cervical disease, 29% had a history of previous conization and 3.7% had a history of trachelectomy. The sensitivity, specificity, negative predictive value, and positive predictive value of the VITOM for cervical intraepithelial neoplasia 2, 3 were 90%, 77%, 90% and 77%, respectively. Concordance of exocolposcopic impressions and histologic results was higher in high-grade lesions (K = 0.68, 95% CI = 0.32-0.87, p < .001) than in low-grade lesions (K = 0.41, 95% CI = 0.1-0.41, p < .05)., Conclusions: : Exocolposcopy with the VITOM is accurate and shows good correlation to histologic findings in high-grade disease of the lower genital tract. The potential advantages include patient and trainee involvement in examination, decision making, and documentation.

Limited sample size and low sensitivity of currently used functional assays challenge direct analysis of cytotoxic CD8+ T lymphocyte activity to quantify antigen-specific immunity after infection or vaccination. Our flow cytometry-based assay reproducibly detects at least three epitope-specific CD8+ T lymphocytes by their cytolytic function. As exemplified for viral epitopes restricted to the human leukocyte antigen (HLA)-A2, the HLA-A2+ human somatic cell hybrid T2 provided an about 10-fold more sensitive readout as compared to autologous B-lymphoblastoid cells or the human erythroleukemia cell line K562 transfected to express HLA-A2 when used as target cells. We named our assay VITAL-FR assay, referring to Hermans et al. (2004) and indicating the modification of using Far Red (FR) dye instead of CMTMR. Under optimal conditions the VITAL-FR assay proved 30 times more sensitive than the 51chromium-release assay to assess epitope-specific target cell lysis. The high overall sensitivity of the VITAL-FR assay basically depended on the negligible spectral overlap of the emission of a stable Far Red fluorescent reporter with the green tracer for target cell labelling. It also profited from long co-incubation of effector and target cells of up to 72, from prior in-vitro culture increasing the frequency of epitope-specific CD8+ T cells and from generic, easily accessible standardized target cells that were used with only 10(3) specific and 10(3) control target cells per individual experimental reaction. Our functional approach with the VITAL-FR assay therefore ideally suits for monitoring CD8+ T cell-mediated cytotoxicity in e.g. vaccination studies with known MHC-restricted immunogenic peptides in scientific and diagnostic applications., (2010 Elsevier B.V. All rights reserved.)

Objective: Most of primary human cancer tissues show effective engraftment and proliferation after transplantation onto Scid mice. However xenotransplantation of vital specimens of cervical carcinoma has not been successful in the past, also the generation of cell lines from primary cervical cancer has hardly ever been possible. The lack of appropriate xenograft models impedes the search for improved specific therapeutic agents., Methods: We explored the efficiency of different techniques for tumor transplantation and describe the first protocol to enable reliable and efficient engraftment of human cervical cancer in Scid beige mice. To demonstrate the value of this tumor model, we explored the therapeutic potency of a novel immunotoxin (SA2E). SA2E is a chimeric protein constructed by fusing the human epidermal growth factor and the plant protein toxin saporin., Results: About 70% of transplanted tumors exhibited potent proliferation, and multiple retransplantation was possible in 40%. Local treatment with the immunotoxin SA2E had a dose dependent therapeutic effect and achieved a tumor volume reduction of up to 60%., Conclusions: Reliable engraftment and high reproducibility make this novel xenograft model an attractive test system to identify new therapeutic agents for cervical cancer., (Copyright 2010. Published by Elsevier Inc.)

We generated an adenovirus-based T-cell vaccine (Ad-p14) that reliably elicits T-cell responses to human papillomavirus (HPV) oncogenes of the 2 most common high-risk HPV serotypes. The artificial gene used to create the vaccine comprising 415 aa (1248 bp) was cloned by fusing 14 polymerase chain reaction fragments of HPV16 and HPV18 E6 and E7 oncogenes devoid of sequences with transforming potential. Although ensuring maximal biologic safety, the construct includes approximately 70% of the relevant T-cell epitopes. In a tumor model for cervical cancer (C3), therapeutic vaccination led to complete eradication in 100% of the mice. In a second model (TC1), it induced initial tumor mass reduction, but 90% of the animals showed delayed tumor progression. To further improve the therapeutic effect, vaccination was combined with systemic application of imiquimod, anti-CD4, alpha-interferon, or anti-GITR. Although adding alpha-interferon improved the therapeutic potential of Ad-p14 by 40%, the combination with anti-GITR resulted in complete and permanent eradication of all TC1 tumors. Ad-p14 has clinical potential for treating HPV-induced lesions, and the added effect of immune response modifiers stresses the importance of combined protocols for immunotherapy of malignant tumors.

Regulatory (FOXP3+) T cells (Tregs) comprise a subpopulation of CD4+ T cells that suppress autoreactive immune cells, thereby protecting organs and tissues from autoimmunity. Tregs have also been detected in human malignancies and their depletion or inactivation substantially improves cellular antitumor immunity in preclinical studies. Novel therapeutic strategies for cervical cancer and precancerous cervical intraepithelial neoplasia (CIN) focus on immune-modulatory and cancer vaccination approaches. In this context, the frequency of Tregs in cervical cancer and precancerous CIN could influence therapeutic strategies. We determined the frequency of infiltrating CD4+ and CD8+ T cells as well as FOXP3+ Tregs in high-grade CIN lesions (CIN III) and cervical carcinoma compared to colon carcinoma, skin melanoma, and bronchial carcinoma. We show that human papilloma virus-derived lesions have a significantly higher number of infiltrating lymphocytes and FOXP3+ Tregs compared to three other common tumor entities. In addition we explored the therapeutic effect of agonistic anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein antibodies that, by single systemic application, inactivate Tregs and induce strong intratumoral invasion of CD8+ T cells and complete tumor eradication in 70% of treated animals. The large number of Tregs in human papilloma virus-derived lesions suggests a pivotal role of Tregs for counteracting the host immune response. We therefore regard CIN and cervical cancer as prime targets for new immune-based non-invasive therapies.

Background: Gene therapy of familial hypercholesterolemia (FH) requires successful transfer and lifelong expression of a functional low density lipoprotein receptor (LDLr) gene in the liver. Most of the vector systems currently employed for gene therapy use promoter elements which do not modulate transgene expression in a physiological manner., Methods: To study the in vivo effects of constitutive LDLr gene expression in the absence of interfering immunological reactions we established a new mouse model which combines homozygous LDLr deficiency and severe combined immune deficiency (SCID)., Results: Adenovirus-mediated transfer and expression of the LDLr gene under the control of a commonly used virus-derived promoter (minimal CMV promoter) leads to prolonged reduction of serum cholesterol levels in LDLr-deficient SCID mice. During the first 10 days after gene therapy serum cholesterol drops to about 10% of pretherapeutic values. Serum cholesterol persists on this level for 2 weeks and then slowly starts to rise again. Four months after vector application serum levels have reached about 40% of pretherapeutic values. However, as early as 5 days after gene transfer, the histological analysis of liver sections revealed the formation of crystalline lipid/cholesterol deposits in the cytosol of hepatocytes. During the following 8 weeks the amount of crystals increased in size and density. The intracellular storage of lipid and cholesterol reduced cell viability and induced an accelerated loss of therapeutic DNA from mice livers as was shown in a comparative expression study employing a transgene with a different metabolic function (human alpha 1-antitrypsin)., Conclusions: The non-physiological constitutive overexpression of an LDL receptor gene induces an imbalance between the speed of LDL uptake and metabolism which leads to pathological accumulation of lipids and cholesterol in hepatocytes. To protect cells from negative effects of LDLr overexpression, future vector design should consider the use of physiologically controlled expression elements., (Copyright 2004 John Wiley & Sons, Ltd.)