Efficient Delivery of Human Cytomegalovirus T Cell Antigens by Attenuated Sendai Virus Vectors.

Human cytomegalovirus (HCMV) represents a major cause of clinical complications during pregnancy as well as immunosuppression, and the licensing of a protective HCMV vaccine remains an unmet global need. Here, we designed and validated novel Sendai virus (SeV) vectors delivering the T cell immunogens IE-1 and pp65. To enhance vector safety, we used a replication-deficient strain (rdSeV) that infects target cells in a nonproductive manner while retaining viral gene expression. In this study, we explored the impact that transduction with rdSeV has on human dendritic cells (DCs) by comparing it to the parental, replication-competent Sendai virus strain (rcSeV) as well as the poxvirus strain modified vaccinia Ankara (MVA). We found that wild-type SeV is capable of replicating to high titers in DCs while rdSeV infects cells abortively. Due to the higher degree of attenuation, IE-1 and pp65 protein levels mediated by rdSeV after infection of DCs were markedly reduced compared to those of the parental Sendai virus recombinants, but antigen-specific restimulation of T cell clones was not negatively affected by this. Importantly, rdSeV showed reduced cytotoxic effects compared to rcSeV and MVA and was capable of mediating DC maturation as well as secretion of alpha interferon and interleukin-6. Finally, in a challenge model with a murine cytomegalovirus (MCMV) strain carrying an HCMV pp65 peptide, we found that viral replication was restricted if mice were previously vaccinated with rdSeV-pp65. Taken together, these data demonstrate that rdSeV has great potential as a vector system for the delivery of HCMV immunogens.IMPORTANCE HCMV is a highly prevalent betaherpesvirus that establishes lifelong latency after primary infection. Congenital HCMV infection is the most common viral complication in newborns, causing a number of late sequelae ranging from impaired hearing to mental retardation. At the same time, managing HCMV reactivation during immunosuppression remains a major hurdle in posttransplant care. Since options for the treatment of HCMV infection are still limited, the development of a vaccine to confine HCMV-related morbidities is urgently needed. We generated new vaccine candidates in which the main targets of T cell immunity during natural HCMV infection, IE-1 and pp65, are delivered by a replication-deficient, Sendai virus-based vector system. In addition to classical prophylactic vaccine concepts, these vectors could also be used for therapeutic applications, thereby expanding preexisting immunity in high-risk groups such as transplant recipients or for immunotherapy of glioblastomas expressing HCMV antigens.

A Respiratory Syncytial Virus Vaccine Vectored by a Stable Chimeric and Replication-Deficient Sendai Virus Protects Mice without Inducing Enhanced Disease.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory infections in children and elderly people, and no marketed vaccine exists. In this study, we generated and analyzed a subunit vaccine against RSV based on a novel genome replication-deficient Sendai virus (SeV) vector. We inserted the RSV F protein, known to be a genetically stable antigen, into our vector in a specific way to optimize the vaccine features. By exchanging the ectodomain of the SeV F protein for its counterpart from RSV, we created a chimeric vectored vaccine that contains the RSV F protein as an essential structural component. In this way, the antigen is actively expressed on the surfaces of vaccine particles in its prefusion conformation, and as recently reported for other vectored vaccines, the occurrence of silencing mutations of the transgene in the vaccine genome can be prevented. In addition, its active gene expression contributes to further stimulation of the immune response. In order to understand the best route of immunization, we compared vaccine efficacies after intranasal (i.n.) or intramuscular (i.m.) immunization of BALB/c mice. Via both routes, substantial RSV-specific immune responses were induced, consisting of serum IgG and neutralizing antibodies, as well as cytotoxic T cells. Moreover, i.n. immunization was also able to stimulate specific mucosal IgA in the upper and lower respiratory tract. In virus challenge experiments, animals were protected against RSV infection after both i.n. and i.m. immunization without inducing vaccine-enhanced disease. Above all, the replication-deficient SeV appeared to be safe and well tolerated.IMPORTANCE Respiratory syncytial virus (RSV) is a major cause of respiratory diseases in young children and elderly people worldwide. There is a great demand for a licensed vaccine. Promising existing vaccine approaches based on live-attenuated vaccines or viral vectors have suffered from unforeseen drawbacks related to immunogenicity and attenuation. We provide a novel RSV vaccine concept based on a genome replication-deficient Sendai vector that has many favorable vaccine characteristics. The specific vaccine design guarantees genetic stability of the transgene; furthermore, it supports a favorable presentation of the antigen, activating the adaptive response, features that other vectored vaccine approaches have often had difficulties with. Wide immunological and pathological analyses in mice confirmed the validity and efficacy of this approach after both parenteral and mucosal administration. Above all, this concept is suitable for initiating clinical studies, and it could also be applied to other infectious diseases.

Genotoxic effects of zinc oxide nanoparticles.

The potential toxicity of nanoparticles has currently provoked public and scientific discussions, and attempts to develop generally accepted handling procedures for nanoparticles are under way. The investigation of the impact of nanoparticles on human health is overdue and reliable test systems accounting for the special properties of nanomaterials must be developed. Nanoparticular zinc oxide (ZnO) may be internalised through ambient air or the topical application of cosmetics, only to name a few, with unpredictable health effects. Therefore, we analysed the determinants of ZnO nanoparticle (NP) genotoxicity. ZnO NPs (15-18 nm in diameter) were investigated at concentrations of 0.1, 10 and 100 µg mL(-1) using the cell line A549. Internalised NPs were only infrequently detectable by TEM, but strongly increased Zn(2+) levels in the cytoplasm and even more in the nuclear fraction, as measured by atom absorption spectroscopy, indicative of an internalised zinc and nuclear accumulation. We observed a time and dosage dependent reduction of cellular viability after ZnO NP exposure. ZnCl2 exposure to cells induced similar impairments of cellular viability. Complexation of Zn(2+) with diethylene triamine pentaacetic acid (DTPA) resulted in the loss of toxicity of NPs, indicating the relevant role of Zn(2+) for ZnO NP toxicity. Foci analyses showed the induction of DNA double strand breaks (DSBs) by ZnO NPs and increased intracellular reactive oxygen species (ROS) levels. Treatment of the cells with the ROS scavenger N-acetyl-l-cysteine (NAC) resulted in strongly decreased intracellular ROS levels and reduced DNA damage. However, a slow increase of ROS after ZnO NP exposure and reduced but not quashed DSBs after NAC-treatment suggest that Zn(2+) may exert genotoxic activities without the necessity of preceding ROS-induction. Our data indicate that ZnO NP toxicity is a result of cellular Zn(2+) intake. Subsequently increased ROS-levels cause DNA damage. However, we found evidence for the assumption that DNA-DSBs could be caused by Zn(2+) without the involvement of ROS.

Development of a versatile and stable internal control system for RT-qPCR assays.

RT-qPCR, an established method for the detection of RNA viruses, requires internal RNA controls for the correct interpretation of PCR results. Robust and versatile RT-PCR controls can be achieved for example by packaging RNA into a virus-derived protein shell. In this study a MS2-based internal control system was developed, that allows stable and universal packing of different RNAs into non-infectious, non-lytic MS2-based viral like particles (VLPs). Two competitive internal controls for a hantavirus assay and a Crimean-Congo Hemorrhagic Fever Virus (CCHFV) assay were cloned for the expression of VLPs. The expression of VLPs containing the RNA of interest could be induced with arabinose in Escherichia coli. The VLPs proved to be temperature resistant and could be frozen and thawed several times without degradation. Distinction of IC RNA from the target RNA was facilitated by a clear shift in the melting temperature or by specific hybridization signals. Furthermore, target and IC PCR amplification could be easily distinguished by their size in gel-electrophoretic analyses. Limits of detection were determined, demonstrating that the application of the IC did not reduce the sensitivity of the target RT-qPCR reactions. The system can be adapted to nearly any required sequence, resulting in a highly flexible method with broad range applications.

Simultaneous detection of ricin and abrin DNA by real-time PCR (qPCR).

Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5'-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

Genetic stability of recombinant MVA-BN.

MVA-BN (modified vaccinia Ankara-Bavarian Nordic) is a highly attenuated vaccinia virus, which serves for development of highly immunogenic recombinant vaccines against infectious diseases and cancer. For generation of recombinant vaccines, insertion of two or more genes at one integration site is preferable, because it is less time consuming and labour intensive. The conventional approach of controlling the two inserts by two different promoters may result in different expression levels of the transgenes. In generating a recombinant MVA-BN (recMVA-BN) vaccine expressing HIV genes, we inserted a tat gene and the fusion gene gag-pol, each under the control of the cowpox ATI promoter into one intergenic region (IGR). The IGRs are sites we specifically developed to stably integrate genes of interest. After 20 passages of the recombinant virus under selective conditions, PCR analysis of IGR showed no remaining contamination with empty vector for recMVA-BN. Further PCR analysis results demonstrated that no homologous recombination between the two ATI sequences had occurred. Moreover, sequencing of the inserted genes and surrounding region demonstrated sequence stability, and RT-PCR confirmed transcription of the inserted genes. The results of our investigation clearly show that a stable insertion of multiple genes each carrying the same promoter at one IGR site is possible and that these insertions show neither detectable genetic instability nor alterations in the transcription of the inserted genes even after many passages.