Preferential Expansion of HPV16 E1-Specific T Cells from Healthy Donors' PBMCs after Ex Vivo Immunization with an E1E2E6E7 Fusion Antigen.

Persistent human papillomavirus (HPV) infection is responsible for practically all cervical and a high proportion of anogenital and oropharyngeal cancers. Therapeutic HPV vaccines in clinical development show great promise in improving outcomes for patients who mount an anti-HPV T-cell response; however, far from all patients elicit a sufficient immunological response. This demonstrates a translational gap between animal models and human patients. Here, we investigated the potential of a new assay consisting of co-culturing vaccine-transduced dendritic cells (DCs) with syngeneic, healthy, human peripheral blood mononuclear cells (PBMCs) to mimic a human in vivo immunization. This new promising human ex vivo PBMC assay was evaluated using an innovative therapeutic adenovirus (Adv)-based HPV vaccine encoding the E1, E2, E6, and E7 HPV16 genes. This new method allowed us to show that vaccine-transduced DCs yielded functional effector T cells and unveiled information on immunohierarchy, showing E1-specific T-cell immunodominance over time. We suggest that this assay can be a valuable translational tool to complement the known animal models, not only for HPV therapeutic vaccines, and supports the use of E1 as an immunotherapeutic target. Nevertheless, the findings reported here need to be validated in a larger number of donors and preferably in patient samples.

The Insertion of an Evolutionary Lost Four-Amino-Acid Cytoplasmic Tail Peptide into a Syncytin-1 Vaccine Increases T- and B-Cell Responses in Mice.

Human endogenous retrovirus type W (HERV-W) is expressed in various cancers. We previously developed an adenovirus-vectored cancer vaccine targeting HERV-W by encoding an assembled HERV-W group-specific antigen sequence and the HERV-W envelope sequence Syncytin-1. Syncytin-1 is constitutively fusogenic and forms large multinucleated cell fusions when overexpressed. Consequently, immunising humans with a vaccine encoding Syncytin-1 can lead to the formation of extensive syncytia, which is undesirable and poses a potential safety issue. Here, we show experiments in cell lines that restoring an evolutionary lost cleavage site of the fusion inhibitory R-peptide of Syncytin-1 inhibit cell fusion. Interestingly, this modification of the HERV-W vaccine's fusogenicity increased the expression of the vaccine antigens in vitro. It also enhanced Syncytin-1-specific antibody responses and CD8+-mediated T-cell responses compared to the wildtype vaccine in vaccinated mice, with a notable enhancement in responses to subdominant T-cell epitopes but equal responses to dominant epitopes and similar rates of survival following a tumour challenge. The impairment of cell-cell fusion and the enhanced immunogenicity profile of this HERV-W vaccine strengthens the prospects of obtaining a meaningful immune response against HERV-W in patients with HERV-W-overexpressing cancers.

Human Ad19a/64 HERV-W Vaccines Uncover Immunosuppression Domain-Dependent T-Cell Response Differences in Inbred Mice.

Expression of human endogenous retrovirus type W (HERV-W) has been linked to cancer, making HERV-W antigens potential targets for therapeutic cancer vaccines. In a previous study, we effectively treated established tumours in mice by using adenoviral-vectored vaccines targeting the murine endogenous retrovirus envelope and group-specific antigen (Gag) of melanoma-associated retrovirus (MelARV) in combination with anti-PD-1. To break the immunological tolerance to MelARV, we mutated the immunosuppressive domain (ISD) of the MelARV envelope. However, reports on the immunogenicity of the HERV-W envelope, Syncytin-1, and its ISD are conflicting. To identify the most effective HERV-W cancer vaccine candidate, we evaluated the immunogenicity of vaccines encoding either the wild-type or mutated HERV-W envelope ISD in vitro and in vivo. Here, we show that the wild-type HERV-W vaccine generated higher activation of murine antigen-presenting cells and higher specific T-cell responses than the ISD-mutated counterpart. We also found that the wild-type HERV-W vaccine was sufficient to increase the probability of survival in mice subjected to HERV-W envelope-expressing tumours compared to a control vaccine. These findings provide the foundation for developing a therapeutic cancer vaccine targeting HERV-W-positive cancers in humans.

Efficacy and Synergy with Cisplatin of an Adenovirus Vectored Therapeutic E1E2E6E7 Vaccine against HPV Genome-Positive C3 Cancers in Mice.

Human papillomavirus (HPV) infections are the main cause of cervical and oropharyngeal cancers. As prophylactic vaccines have no curative effect, an efficient therapy would be highly desired. Most therapeutic vaccine candidates target only a small subset of HPV regulatory proteins, namely, E6 and E7, and are therefore restricted in the breadth of their immune response. However, research has suggested E1 and E2 as promising targets to fight HPV+ cancer. Here, we report the design of adenoviral vectors efficiently expressing HPV16 E1 and E2 in addition to transformation-deficient E6 and E7. Vaccination elicited vigorous CD4+ and CD8+ T-cell responses against all encoded HPV16 proteins in outbred mice and against E1 and E7 in C57BL/6 mice. Therapeutic vaccination of C3 tumor-bearing mice led to significantly reduced tumor growth and enhanced survival for both small and established tumors. Tumor biopsies revealed increased numbers of tumor-infiltrating CD8+ T cells in treated mice. Cisplatin enhanced the effect of therapeutic vaccination, accompanied by enhanced infiltration of dendritic cells into the tumor. CD8+ T cells were identified as effector cells in T-cell depletion assays, seemingly under regulation by FoxP3+CD4+ regulatory T cells. Finally, therapeutic vaccination with Ad-Ii-E1E2E6E7 exhibited significantly enhanced survival compared with vaccination with two peptides each harboring a known E6/E7 epitope. We hypothesize that this difference could be due to the induction of additional T-cell responses against E1. These results support the use of this novel vaccine candidate targeting an extended set of antigens (Ad-Ii-E1E2E6E7), in combination with cisplatin, as an advanced strategy to combat HPV+ cancers.

Breaking Entry-and Species Barriers: LentiBOOST® Plus Polybrene Enhances Transduction Efficacy of Dendritic Cells and Monocytes by Adenovirus 5.

Due to their ability to trigger strong immune responses, adenoviruses (HAdVs) in general and the serotype5 (HAdV-5) in particular are amongst the most popular viral vectors in research and clinical application. However, efficient transduction using HAdV-5 is predominantly achieved in coxsackie and adenovirus receptor (CAR)-positive cells. In the present study, we used the transduction enhancer LentiBOOST® comprising the polycationic Polybrene to overcome these limitations. Using LentiBOOST®/Polybrene, we yielded transduction rates higher than 50% in murine bone marrow-derived dendritic cells (BMDCs), while maintaining their cytokine expression profile and their capability to induce T-cell proliferation. In human dendritic cells (DCs), we increased the transduction rate from 22% in immature (i)DCs or 43% in mature (m)DCs to more than 80%, without inducing cytotoxicity. While expression of specific maturation markers was slightly upregulated using LentiBOOST®/Polybrene on iDCs, no effect on mDC phenotype or function was observed. Moreover, we achieved efficient HAdV5 transduction also in human monocytes and were able to subsequently differentiate them into proper iDCs and functional mDCs. In summary, we introduce LentiBOOST® comprising Polybrene as a highly potent adenoviral transduction agent for new in-vitro applications in a set of different immune cells in both mice and humans.

Design and Immunological Validation of Macaca fascicularis Papillomavirus Type 3 Based Vaccine Candidates in Outbred Mice: Basis for Future Testing of a Therapeutic Papillomavirus Vaccine in NHPs.

Persistent human papillomavirus (HPV) infections are causative for cervical neoplasia and carcinomas. Despite the availability of prophylactic vaccines, morbidity and mortality induced by HPV are still too high. Thus, an efficient therapy, such as a therapeutic vaccine, is urgently required. Herein, we describe the development and validation of Macaca fascicularis papillomavirus type 3 (MfPV3) antigens delivered via nucleic-acid and adenoviral vectors in outbred mouse models. Ten artificially fused polypeptides comprising early viral regulatory proteins were designed and optionally linked to the T cell adjuvant MHC-II-associated invariant chain. Transfected HEK293 cells and A549 cells transduced with recombinant adenoviruses expressing the same panel of artificial antigens proved proper and comparable expression, respectively. Immunization of outbred CD1 and OF1 mice led to CD8+ and CD4+ T cell responses against MfPV3 antigens after DNA- and adenoviral vector delivery. Moreover, in vivo cytotoxicity of vaccine-induced CD8+ T cells was demonstrated in BALB/c mice by quantifying specific killing of transferred peptide-pulsed syngeneic target cells. The use of the invariant chain as T cell adjuvant enhanced the T cell responses regarding cytotoxicity and in vitro analysis suggested an accelerated turnover of the antigens as causative. Notably, the fusion-polypeptide elicited the same level of T-cell responses as administration of the antigens individually, suggesting no loss of immunogenicity by fusing multiple proteins in one vaccine construct. These data support further development of the vaccine candidates in a follow up efficacy study in persistently infected Macaca fascicularis monkeys to assess their potential to eliminate pre-malignant papillomavirus infections, eventually instructing the design of an analogous therapeutic HPV vaccine.

Differential upregulation of host cell protein kinases by the replication of α-, ß- and γ-herpesviruses provides a signature of virus-specific signalling.

Infections with human herpesviruses share several molecular characteristics, but the diversified medical outcomes are distinct to viral subfamilies and species. Notably, both clinical and molecular correlates of infection are a challenging field and distinct patterns of virus-host interaction have rarely been defined; this study therefore focuses on the search for virus-specific molecular indicators. As previous studies have demonstrated the impact of herpesvirus infections on changes in host signalling pathways, we illustrate virus-modulated expression levels of individual cellular protein kinases. Current data reveal (i) α-, ß- and γ-herpesvirus-specific patterns of kinase modulation as well as (ii) differential levels of up-/downregulated kinase expression and phosphorylation, which collectively suggest (iii) defined signalling patterns specific for the various viruses (VSS) that may prove useful for defining molecular indicators. Combined, the study confirms the correlation between herpesviral replication and modulation of signalling kinases, possibly exploitable for the in vitro characterization of viral infections.

Improving Lentiviral Transduction of CD34+ Hematopoietic Stem and Progenitor Cells.

The delivery of therapeutic genes for treatment of inherited or infectious diseases frequently requires lentiviral transduction of CD34+ hematopoietic stem and progenitor cells (HSC). Optimized transduction protocols with a therapeutic goal aim to maximize the number of transduction-positive cells while limiting the vector copy number that reach each individual cell. Importantly, the transduced HSC should maintain their "stem-like" properties. Here, we analyzed LentiBOOST™ reagent, a membrane-sealing poloxamer, with respect to enhancing lentiviral transduction of CD34+ peripheral blood stem cells. We demonstrate that inclusion of LentiBOOST™ in a standard HSC transduction protocol yields high transduction efficiencies while preserving the ability of the transduced HSC to differentiate into various hematopoietic lineages. Thus, LentiBOOST™ reagent can significantly improve lentiviral CD34+ HSC transduction protocols with the potential to improve production of gene-modified cell products.

Vaccine vectors based on Adenovirus 19a/64 exhibit broad cellular tropism and potently restimulate HCMV-specific T cell responses ex vivo.

Human Cytomegalovirus (HCMV) remains a major health burden and the development of a vaccine is a global priority. We developed new viral vectors delivering the T cell immunogens IE-1 and pp65 based on Adenovirus 19a/64 (Ad19a/64), a member of subgroup D. In this ex vivo study, the novel vectors were compared side by side to Ad5 or modified Vaccinia Ankara (MVA) strains expressing the same transgenes. We found that unlike Ad5, Ad19a/64 vectors readily transduce a broad panel of immune cells, including monocytes, T cells, NK cells and monocyte-derived dendritic cells (moDCs). Both Ad19a/64- and MVA-transduced moDCs efficiently restimulated IE-1 or pp65-specific T cells but MVA induced a higher amount of cytotoxicity in this cell type. Ad5 and Ad19 induced upregulation of CD86 and HLA-DR in moDCs whereas expression of CD80 and CD83 was largely unaltered. By contrast, MVA transduction led to downregulation of all markers. Taken together, our data demonstrate that Ad19a/64 is a promising vector for the delivery of HCMV immunogens since it transduces dendritic cells with an efficiency that is comparable to MVA, but cytotoxicity and interference with dendritic cell maturation are less pronounced.

Versatile Viral Vector Strategies for Postscreening Target Validation and RNAi ON-Target Control.

Our approach aims to optimize postscreening target validation strategies using viral vector-driven RNA interference (RNAi) cell models. The RNAiONE validation platform is an array of plasmid-based expression vectors that each drives tandem expression of the gene of interest (GOI) with one small hairpin RNA (shRNA) from a set of computed candidate sequences. The best-performing shRNA (>85% silencing efficiency) is then integrated in an inducible, all-in-one lentiviral vector to transduce pharmacologically relevant cell types that endogenously express the GOI. VariCHECK is used subsequently to combine the inducible knockdown with an equally inducible rescue of the GOI for ON-target phenotype verification. The complete RNAiONE-VariCHECK system relies on three key elements to ensure high predictability: (1) maximized silencing efficiencies by a focused shRNA validation process, (2) homogeneity of the RNAi cell pools by application of sophisticated viral vector technologies, and (3) exploiting the advantages of inducible expression systems. By using a reversible expression system, our strategy adds critical information to hot candidates from RNAi screens and avoids potential side effects that may be caused by other, irreversible genomic manipulation methods such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas). This approach will add credibility to top-hit screening candidates and protect researchers from costly misinterpretations early in the preclinical drug development process.