A Novel Orf Virus D1701-VrV-Based Dengue Virus (DENV) Vaccine Candidate Expressing HLA-Specific T Cell Epitopes: A Proof-of-Concept Study.

Although dengue virus (DENV) affects almost half of the world's population there are neither preventive treatments nor any long-lasting and protective vaccines available at this time. The complexity of the protective immune response to DENV is still not fully understood. The most advanced vaccine candidates focus specifically on humoral immune responses and the production of virus-neutralizing antibodies. However, results from several recent studies have revealed the protective role of T cells in the immune response to DENV. Hence, in this study, we generated a novel and potent DENV vaccine candidate based on an Orf virus (ORFV, genus Parapoxvirus) vector platform engineered to encode five highly conserved or cross-reactive DENV human leukocyte antigen (HLA)-A*02- or HLA-B*07-restricted epitopes as minigenes (ORFV-DENV). We showed that ORFV-DENV facilitates the in vitro priming of CD8+ T cells from healthy blood donors based on responses to each of the encoded immunogenic peptides. Moreover, we demonstrated that peripheral blood mononuclear cells isolated from clinically confirmed DENV-positive donors stimulated with ORFV-DENV generate cytotoxic T cell responses to at least three of the expressed DENV peptides. Finally, we showed that ORFV-DENV could activate CD8+ T cells isolated from donors who had recovered from Zika virus (ZIKV) infection. ZIKV belongs to the same virus family (Flaviviridae) and has epitope sequences that are homologous to those of DENV. We found that highly conserved HLA-B*07-restricted ZIKV and DENV epitopes induced functional CD8+ T cell responses in PBMCs isolated from confirmed ZIKV-positive donors. In summary, this proof-of-concept study characterizes a promising new ORFV D1701-VrV-based DENV vaccine candidate that induces broad and functional epitope-specific CD8+ T cell responses.

Genomic Characterization of Orf Virus Strain D1701-V (Parapoxvirus) and Development of Novel Sites for Multiple Transgene Expression.

The Orf virus (ORFV; Parapoxvirus) strain D1701 with an attenuated phenotype and excellent immunogenic capacity is successfully used for the generation of recombinant vaccines against different viral infections. Adaption for growth in Vero cells was accompanied by additional major genomic changes resulting in ORFV strain variant D1701-V. In this study, restriction enzyme mapping, blot hybridization and DNA sequencing of the deleted region s (A, AT and D) in comparison to the predecessor strain D1701-B revealed the loss of 7 open reading frames (ORF008, ORF101, ORF102, ORF114, ORF115, ORF116, ORF117). The suitability of deletion site D for expression of foreign genes is demonstrated using novel synthetic early promoter eP1 and eP2. Comparison of promoter strength showed that the original vegf-e promoter Pv as well as promoter eP2 display an up to 11-fold stronger expression than promoter eP1, irrespective of the insertion site. Successful integration and expression of the fluorescent marker genes is demonstrated by gene- and insertion-site specific PCR assays, fluorescence microscopy and flow cytometry. For the first time ORFV recombinants are generated simultaneously expressing transgenes in two different insertion loci. That allows production of polyvalent vaccines containing several antigens against one or different pathogens in a single vectored ORFV vaccine.

Wall Teichoic Acid Glycosylation Governs Staphylococcus aureus Nasal Colonization.

UNLABELLED: Nasal colonization by the human pathogen Staphylococcus aureus is a major risk factor for hospital- and community-acquired infections. A key factor required for nasal colonization is a cell surface-exposed zwitterionic glycopolymer, termed wall teichoic acid (WTA). However, the precise mechanisms that govern WTA-mediated nasal colonization have remained elusive. Here, we report that WTA GlcNAcylation is a pivotal requirement for WTA-dependent attachment of community-acquired methicillin-resistant S. aureus (MRSA) and emerging livestock-associated MRSA to human nasal epithelial cells, even under conditions simulating the nutrient composition and dynamic flow of nasal secretions. Depending on the S. aureus strain, WTA O-GlcNAcylation occurs in either α or ß configuration, which have similar capacities to mediate attachment to human nasal epithelial cells, suggesting that many S. aureus strains maintain redundant pathways to ensure appropriate WTA glycosylation. Strikingly, a lack of WTA glycosylation significantly abrogated the ability of MRSA to colonize cotton rat nares in vivo. These results indicate that WTA glycosylation modulates S. aureus nasal colonization and may help to develop new strategies for eradicating S. aureus nasal colonization in the future. IMPORTANCE: Nasal colonization by the major human pathogen Staphylococcus aureus is a risk factor for severe endogenous infections and contributes to the spread of this microbe in hospitals and the community. Here, we show that wall teichoic acid (WTA) O-GlcNAcylation is a key factor required for S. aureus nasal colonization. These data provide a mechanistic explanation for the capacity of WTA to modulate S. aureus nasal colonization and may stimulate research activities to establish valuable strategies to eradicate S. aureus nasal colonization in high-risk hospitalized patients and in the general community.

Temporal vocal features suggest different call-pattern generating mechanisms in mice and bats.

BACKGROUND: Mice produce ultrasonic vocalizations in various inter-individual encounters and with high call rates. However, it is so far virtually unknown how these vocal patterns are generated. On the one hand, these vocal patterns could be embedded into the normal respiratory cycle, as happens in bats and other mammals that produce similar call rates and frequencies. On the other, mice could possess distinct vocal pattern generating systems that are capable of modulating the respiratory cycle, which is what happens in non-human and human primates. In the present study, we investigated the temporal call patterns of two different mammalian species, bats and mice, in order to differentiate between these two possibilities for mouse vocalizations. Our primary focus was on comparing the mechanisms for the production of rapid, successive ultrasound calls of comparable frequency ranges in the two species. RESULTS: We analyzed the temporal call pattern characteristics of mice, and we compared these characteristics to those of ultrasonic echolocation calls produced by horseshoe bats. We measured the distributions of call durations, call intervals, and inter-call intervals in the two species. In the bat, and consistent with previous studies, we found that call duration was independent of corresponding call intervals, and that it was negatively correlated with the corresponding inter-call interval. This indicates that echolocation call production mechanisms in the bat are highly correlated with the respiratory cycle. In contrast, call intervals in the mouse were directly correlated with call duration. Importantly, call duration was not, or was only slightly, correlated with inter-call intervals, consistent with the idea that vocal production in the mouse is largely independent of the respiratory cycle. CONCLUSIONS: Our findings suggest that ultrasonic vocalizations in mice are produced by call-pattern generating mechanisms that seem to be similar to those that have been found in primates. This is in contrast to the production mechanisms of ultrasonic echolocation calls in horseshoe bats. These results are particularly interesting, especially since mouse vocalizations have recently attracted increased attention as potential indicators for the degree of progression of several disease patterns in mouse models for neurodegenerative and neurodevelopmental disorders of humans.