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

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

Abstract 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

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

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

Differential upregulation of host cell protein kinases by the replication of α-, β- 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 path- ways, we illustrate virus-modulated expression levels of individual cellular protein kinases. Current data reveal (i) alpha- and beta-, gamma-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