The vaccinia-based Sementis Copenhagen Vector coronavirus disease 2019 vaccine induces broad and durable cellular and humoral immune responses.

The ongoing coronavirus disease 2019 (COVID-19) pandemic perpetuated by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has highlighted the continued need for broadly protective vaccines that elicit robust and durable protection. Here, the vaccinia virus-based, replication-defective Sementis Copenhagen Vector (SCV) was used to develop a first-generation COVID-19 vaccine encoding the spike glycoprotein (SCV-S). Vaccination of mice rapidly induced polyfunctional CD8 T cells with cytotoxic activity and robust type 1 T helper-biased, spike-specific antibodies, which are significantly increased following a second vaccination, and contained neutralizing activity against the alpha and beta variants of concern. Longitudinal studies indicated that neutralizing antibody activity was maintained up to 9 months after vaccination in both young and middle-aged mice, with durable immune memory evident even in the presence of pre-existing vector immunity. Therefore, SCV-S vaccination has a positive immunogenicity profile, with potential to expand protection generated by current vaccines in a heterologous boost format and presents a solid basis for second-generation SCV-based COVID-19 vaccine candidates incorporating additional SARS-CoV-2 immunogens.

Production of a Chikungunya Vaccine Using a CHO Cell and Attenuated Viral-Based Platform Technology.

Vaccinia-based systems have been extensively explored for the development of recombinant vaccines. Herein we describe an innovative vaccinia virus (VACV)-derived vaccine platform technology termed Sementis Copenhagen Vector (SCV), which was rendered multiplication-defective by targeted deletion of the essential viral assembly gene D13L. A SCV cell substrate line was developed for SCV vaccine production by engineering CHO cells to express D13 and the VACV host-range factor CP77, because CHO cells are routinely used for manufacture of biologics. To illustrate the utility of the platform technology, a SCV vaccine against chikungunya virus (SCV-CHIK) was developed and shown to be multiplication-defective in a range of human cell lines and in immunocompromised mice. A single vaccination of mice with SCV-CHIK induced antibody responses specific for chikungunya virus (CHIKV) that were similar to those raised following vaccination with a replication-competent VACV-CHIK and able to neutralize CHIKV. Vaccination also provided protection against CHIKV challenge, preventing both viremia and arthritis. Moreover, SCV retained capacity as an effective mouse smallpox vaccine. In summary, SCV represents a new and safe vaccine platform technology that can be manufactured in modified CHO cells, with pre-clinical evaluation illustrating utility for CHIKV vaccine design and construction.

NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling.

Autophagy is closely related to the occurrence and development of human malignancies; however, the detailed mechanisms underlying autophagy in cervical cancer require further investigation. Previously, we found that the ectopic expression of NCAPH, a regulatory subunit of condensed protein complexes, significantly enhanced the proliferation of tumor cells; however, the underlying mechanisms were unclear. Here, we revealed that NCAPH is a novel autophagy-associated protein in cervical cancer that promotes cell proliferation by inhibiting autophagosome formation and reducing autophagy, with no effect on the cell cycle, apoptosis, or aging. Tripartite motif-containing protein 21 (TRIM21) is well known to be involved in inflammation, autoimmunity and cancer, mainly via its E3 ubiquitin ligase activity. Mass spectrometry and immunoprecipitation assays showed that TRIM21 interacted with NCAPH and decreased the protein stability of NCAPH via ubiquitination at the K11 lysine residue. Structural domain mutation analysis revealed that TRIM21 combined with NCAPH through its PRY/SPRY and CC domains and accelerated the degradation of NCAPH through the RING domain. Furthermore, TRIM21 promoted autophagosome formation and reduced cell proliferation by inhibiting NCAPH expression and the downstream AKT/mTOR pathway in cervical cancer cells. Immunohistochemical staining revealed that the protein expression of TRIM21 was negatively correlated with that of NCAPH and positively correlated with that of beclin-1 in cervical cancer tissues. Therefore, we provide evidence for the role of the TRIM21-NCAPH axis in cervical cancer autophagy and proliferation and the involvement of the AKT/mTOR signaling pathway in this process. These results deepen our understanding of the carcinogenesis of cervical cancer, broaden the understanding of the molecular mechanisms of TRIM21 and NCAPH, and provide guidance for individualized treatment of cervical cancer in the future.

Exposure of insect cells to ionising radiation in vivo induces persistent phosphorylation of a H2AX homologue (H2AvB).

The response of eukaryotic cells to ionising radiation (IR)-induced double-strand DNA breaks is highly conserved and involves a DNA repair mechanism characterised by the early phosphorylation of histone protein H2AX (producing the active form γH2AX). Although the expression of an induced γH2AX variant has been detected in Drosophila melanogaster, the expression and radiation response of a γH2AX homologue has not been reported in economically important fruit flies. We use Bactrocera tryoni (Diptera: Tephritidae, Queensland fruit fly or 'Q-fly') to investigate this response with a view to developing molecular assays to detect/quantify exposure of fruit flies to IR and consequent DNA damage. Deep sequencing confirmed the presence of a H2AX homologue that we have termed H2AvB (i.e. variant Bactrocera) and has an identical sequence to a histone reported from the human disease vector Glossina morsitans. A linear dose-response of γH2AvB (0-400 Gy IR) was observed in whole Q-fly pupal lysates 24h post-IR and was detected at doses as low as 20 Gy. γH2AvB signal peaked at ~20min after IR exposure and at 24h post-IR the signal remained elevated but declined significantly by 5 days. Persistent and dose-dependent γH2AvB signal could be detected and quantified either by western blot or by laser scanning cytometry up to 17 days post-IR exposure in histone extracts or isolated nuclei from adult Q-flies (irradiated as pupae). We conclude that IR exposure in Q-fly leads to persistent γH2AvB signals (over a period of days) that can easily be detected by western blot or quantitative immunofluorescence techniques. These approaches have potential as the basis for assays for detection and quantification of prior IR exposure in pest fruit flies.

Identification of an in vitro interaction between an insect immune suppressor protein (CrV2) and G alpha proteins.

The protein CrV2 is encoded by a polydnavirus integrated into the genome of the endoparasitoid Cotesia rubecula (Hymenoptera:Braconidae:Microgastrinae) and is expressed in host larvae with other gene products of the polydnavirus to allow successful development of the parasitoid. CrV2 expression has previously been associated with immune suppression, although the molecular basis for this was not known. Here, we have used time-resolved Förster resonance energy transfer (TR-FRET) to demonstrate high affinity binding of CrV2 to Gα subunits (but not the Gßγ dimer) of heterotrimeric G-proteins. Signals up to 5-fold above background were generated, and an apparent dissociation constant of 6.2 nm was calculated. Protease treatment abolished the TR-FRET signal, and the presence of unlabeled CrV2 or Gα proteins also reduced the TR-FRET signal. The activation state of the Gα subunit was altered with aluminum fluoride, and this decreased the affinity of the interaction with CrV2. It was also demonstrated that CrV2 preferentially bound to Drosophila Gα(o) compared with rat Gα(i1). In addition, three CrV2 homologs were detected in sequences derived from polydnaviruses from Cotesia plutellae and Cotesia congregata (including the immune-related early expressed transcript, EP2). These data suggest a potential mode-of-action of immune suppressors not previously reported, which in addition to furthering our understanding of insect immunity may have practical benefits such as facilitating development of novel controls for pest insect species.

Systemic mucormycosis in a hooded seal (Cystophora cristata).

A 4-yr-old male captive hooded seal (Cystophora cristata), previously diagnosed as hypothyroid, died after a 3-wk period of lethargy and inappetance despite treatment that included intramuscular administration of antibiotics and multivitamins. Gross pathologic findings included extensive muscle necrosis over the left flank, an underlying necrotic iliac lymph node, two necrotic pulmonary masses and a necrotic bronchial lymph node. Routine cultures yielded a number of bacterial isolates and a heavy pure fungal growth from the necrotic iliac lymph node; wet preparations of which revealed sporangiophores typical of Mucor sp. Histopathology of necrotic muscle, pulmonary lesions and bronchial and iliac lymph nodes revealed necrosis with a marked pyogranulomatous and eosinophilic inflammatory cell infiltrate and fungal hyphae consistent with a Zygomycete species. This is believed to be the first report of systemic mucormycosis in a pinniped likely to have originated from an injection site reaction.

HPV E7-mediated NCAPH ectopic expression regulates the carcinogenesis of cervical carcinoma via PI3K/AKT/SGK pathway.

Cervical cancer is one of the most common gynecological tumors in the world, and human papillomavirus (HPV) infection is its causative agent. However, the molecular mechanisms involved in the carcinogenesis of cervical cancer still require clarification. Here we found that knockdown of Non-SMC (Structural Maintenance of Chromosomes) condensin I complex subunit H (NCAPH) gene expression significantly inhibited the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of cervical cancer cells in vitro, and restrained xenograft tumor formation in vivo. Intriguingly, HPV E7 could form a positive feedback loop with NCAPH. E7 upregulated NCAPH gene expression via E2F1 which initiated NCAPH transcription by binding to its promoter directly. Silencing of NCAPH reduced E7 transcription via promoting the transition of AP-1 heterodimer from c-Fos/c-Jun to Fra-1/c-Jun. Moreover, the E7-mediated NCAPH overexpression was involved in the activation of the PI3K/AKT/SGK signaling pathway. In vivo, NCAPH expression in cervical cancer tissues was significantly higher than which in normal cervix and high-grade squamous intraepithelial lesion (HSIL) tissues, and its expression was significantly correlated with tumor size, depth of invasion and lymph node metastasis. Patients with high NCAPH expression had a significantly better survival outcomes than those with low-expression, suggesting that NCAPH-induced cell proliferation might sensitize cancer cells to adjuvant therapy. In conclusion, our results revealed the role of NCAPH in the carcinogenesis of cervical cancer in vitro and in vivo. The interaction between E7 and NCAPH expands the mechanism of HPV induced tumorigenesis and that of host genes regulating HPV E7.

The vaccinia virus based Sementis Copenhagen Vector vaccine against Zika and chikungunya is immunogenic in non-human primates.

The Sementis Copenhagen Vector (SCV) is a new vaccinia virus-derived, multiplication-defective, vaccine technology assessed herein in non-human primates. Indian rhesus macaques (Macaca mulatta) were vaccinated with a multi-pathogen recombinant SCV vaccine encoding the structural polyproteins of both Zika virus (ZIKV) and chikungunya virus (CHIKV). After one vaccination, neutralising antibody responses to ZIKV and four strains of CHIKV, representative of distinct viral genotypes, were generated. A second vaccination resulted in significant boosting of neutralising antibody responses to ZIKV and CHIKV. Following challenge with ZIKV, SCV-ZIKA/CHIK-vaccinated animals showed significant reductions in viremias compared with animals that had received a control SCV vaccine. Two SCV vaccinations also generated neutralising and IgG ELISA antibody responses to vaccinia virus. These results demonstrate effective induction of immunity in non-human primates by a recombinant SCV vaccine and illustrates the utility of SCV as a multi-disease vaccine platform capable of delivering multiple large immunogens.

Arthritogenic Alphavirus Vaccines: Serogrouping Versus Cross-Protection in Mouse Models.

Chikungunya virus (CHIKV), Ross River virus (RRV), o'nyong nyong virus (ONNV), Mayaro virus (MAYV) and Getah virus (GETV) represent arthritogenic alphaviruses belonging to the Semliki Forest virus antigenic complex. Antibodies raised against one of these viruses can cross-react with other serogroup members, suggesting that, for instance, a CHIKV vaccine (deemed commercially viable) might provide cross-protection against antigenically related alphaviruses. Herein we use human alphavirus isolates (including a new human RRV isolate) and wild-type mice to explore whether infection with one virus leads to cross-protection against viremia after challenge with other members of the antigenic complex. Persistently infected Rag1-/- mice were also used to assess the cross-protective capacity of convalescent CHIKV serum. We also assessed the ability of a recombinant poxvirus-based CHIKV vaccine and a commercially available formalin-fixed, whole-virus GETV vaccine to induce cross-protective responses. Although cross-protection and/or cross-reactivity were clearly evident, they were not universal and were often suboptimal. Even for the more closely related viruses (e.g., CHIKV and ONNV, or RRV and GETV), vaccine-mediated neutralization and/or protection against the intended homologous target was significantly more effective than cross-neutralization and/or cross-protection against the heterologous virus. Effective vaccine-mediated cross-protection would thus likely require a higher dose and/or more vaccinations, which is likely to be unattractive to regulators and vaccine manufacturers.

[35S]GTPgammaS binding in G protein-coupled receptor assays.

The [(35)S]GTPgammaS binding assay to measure G protein activation following agonist binding to G protein-coupled receptors (GPCRs) remains a powerful molecular technique to substantiate traditional pharmacological values of potency, efficacy, and affinity. The method described uses membrane preparations of the alpha(2A)-adrenergic receptor and purified G protein subunits expressed in Sf9 cells, reconstituted into a functional signaling system. This technology is generic and could be used with other GPCRs to demonstrate initial signaling events following receptor activation. Agonist-stimulated [(35)S]GTPgammaS binding is measured in a 96-well plate format using scintillation counting.

A vaccinia-based single vector construct multi-pathogen vaccine protects against both Zika and chikungunya viruses.

Zika and chikungunya viruses have caused major epidemics and are transmitted by Aedes aegypti and/or Aedes albopictus mosquitoes. The "Sementis Copenhagen Vector" (SCV) system is a recently developed vaccinia-based, multiplication-defective, vaccine vector technology that allows manufacture in modified CHO cells. Herein we describe a single-vector construct SCV vaccine that encodes the structural polyprotein cassettes of both Zika and chikungunya viruses from different loci. A single vaccination of mice induces neutralizing antibodies to both viruses in wild-type and IFNAR-/- mice and protects against (i) chikungunya virus viremia and arthritis in wild-type mice, (ii) Zika virus viremia and fetal/placental infection in female IFNAR-/- mice, and (iii) Zika virus viremia and testes infection and pathology in male IFNAR-/- mice. To our knowledge this represents the first single-vector construct, multi-pathogen vaccine encoding large polyproteins, and offers both simplified manufacturing and formulation, and reduced "shot burden" for these often co-circulating arboviruses.

Transient dominant host-range selection using Chinese hamster ovary cells to generate marker-free recombinant viral vectors from vaccinia virus.

Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selection drugs or detection methods, and (ii) dominant host-range selection. The latter uses VACV variants rendered replication-incompetent in host cell lines by the deletion of host-range genes. Replicative ability is restored by co-insertion of the host-range genes, providing for dominant selection of the recombinant viruses. Here, we describe a new method for the construction of rVACVs using the cowpox CP77 protein and unmodified VACV as the starting material. Our selection system will expand the range of tools available for positive selection of rVACV during vector construction, and it is substantially more high-fidelity than approaches based on selection for drug resistance.

Prevalence of Presenting Conditions in Grey Seal Pups (Halichoerus grypus) Admitted for Rehabilitation.

A retrospective survey was performed on the presenting conditions of 205 live grey seal pups (Halichoerus grypus) admitted to the Cornish Seal Sanctuary in Gweek, United Kingdom between May 2005 and March 2011. The purpose of the survey was to examine the prevalence of various presenting signs at the sanctuary. The presenting signs were classified into nine non-mutually exclusive categories: ocular disorders, nasal disorders, oral disorders, respiratory disorders, orthopaedic disorders, puncture wounds, abrasions, netting injuries, and onychia. The sex ratio of seal pups in this study was 1.35 males per female. Of the 205 examined for rehabilitation, 22 (10.73%) did not survive to release. 68.78% of grey seal pups presented with puncture wounds, 47.80% with respiratory disorders, 46.34% with ocular disorders, 42.63% malnourished, 36.59% with abrasions, 25.37% with oral disorders, 23.90% with nasal disorders, 11.71% with orthopaedic disorders, 9.27% with onychia, and 3.41% presented with netting injuries. 52% were normothermic, 42% were hyperthermic, and 5% were hypothermic. Associations between gender, outcome of rehabilitation, hospitalisation time and presenting disorders were examined. In addition, admissions rates were found to display seasonality. The results of this study will aid in future preparation of grey seal rehabilitation facilities.

From crescent to mature virion: vaccinia virus assembly and maturation.

Vaccinia virus (VACV) has achieved unprecedented success as a live viral vaccine for smallpox which mitigated eradication of the disease. Vaccinia virus has a complex virion morphology and recent advances have been made to answer some of the key outstanding questions, in particular, the origin and biogenesis of the virion membrane, the transformation from immature virion (IV) to mature virus (MV), and the role of several novel genes, which were previously uncharacterized, but have now been shown to be essential for VACV virion formation. This new knowledge will undoubtedly contribute to the rational design of safe, immunogenic vaccine candidates, or effective antivirals in the future. This review endeavors to provide an update on our current knowledge of the VACV maturation processes with a specific focus on the initiation of VACV replication through to the formation of mature virions.

Measurement of heterotrimeric G-protein and regulators of G-protein signaling interactions by time-resolved fluorescence resonance energy transfer.

G-protein-coupled receptors transduce their signals through G-protein subunits which in turn are subject to modulation by other intracellular proteins such as the regulators of G-protein signaling (RGS) proteins. We have developed a cell-free, homogeneous (mix and read format), time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor heterotrimeric G-protein subunit interactions and the interaction of the G alpha subunit with RGS4. The assay uses a FRET pair consisting of a terbium cryptate chelate donor spectrally matched to an Alexa546 fluor acceptor, each of which is conjugated to separate protein binding partners, these being G alpha(i1):beta4gamma2 or G alpha(i1):RGS4. Under conditions favoring specific binding between labeled partners, high-affinity interactions were observed as a rapid increase (>fivefold) in the FRET signal. The specificity of these interactions was demonstrated using denaturing or competitive conditions which caused significant reductions in fluorescence (50-85%) indicating that labeled proteins were no longer in close proximity. We also report differential binding effects as a result of altered activation state of the G alpha(i1) protein. This assay confirms that interactions between G-protein subunits and RGS4 can be measured using TR-FRET in a cell- and receptor-free environment.