Travelling with Dengue: From the Skin to the Nodes

Dengue virus (DENV) infects humans through the skin. The early infection and encounters between DENV and cutaneous immune and non‐immune cells only recently are under investigation. We have reported DENV‐infected cutaneous dendritic cells (DCs), also keratinocytes and dermal fibroblasts permissive to DENV infection. Now, upon cutaneously inoculating fluorescently labeled DENV into immune‐competent mice, we found DENV mostly in dermis from 1 h post‐inoculation. Afterwards, DENV rapidly localized in the subcapsular sinus of draining lymph nodes (DLNs) associated with CD169+ macrophages, suggesting virus travelling through lymph flow. However, DENV association with CD11c+ DCs in the paracortex and T:B border suggests DENV being ferried from the skin to DLNs by DCs too. DENV was not associated with F4/80+ macrophages nor with DEC205+ DCs, but it was inside B cell follicles early after cutaneous inoculation. DENV inside B follicles likely affects the development of humoral responses. Antibody responses deserve very careful scrutiny as neutralizing memory antibodies are crucial to counteract homotypic reinfections whereas non‐neutralizing ones might facilitate heterotypic DENV infection or even Zika infection, another flavivirus. Unravelling the DENV journey from skin to lymph into regional nodes and the cellular compartments will aid to understand the disease, its pathology and how to counteract it

Crosstalk between dermal fibroblasts and dendritic cells during dengue virus infection

Dengue virus infection (DENV-2) is transmitted by infected mosquitoes via the skin, where many dermal and epidermal cells are potentially susceptible to infection. Most of the cells in an area of infection will establish an antiviral microenvironment to control viral replication. Although cumulative studies report permissive DENV-2 infection in dendritic cells, keratinocytes, and fibroblasts, among other cells also infected, little information is available regarding cell-to-cell crosstalk and the effect of this on the outcome of the infection. Therefore, our study focused on understanding the contribution of fibroblast and dendritic cell crosstalk to the control or promotion of dengue. Our results suggest that dendritic cells promote an antiviral state over fibroblasts by enhancing the production of type I interferon, but not proinflammatory cytokines. Infected and non-infected fibroblasts promoted partial dendritic cell maturation, and the fibroblast-matured cells were less permissive to infection and showed enhanced type I interferon production. We also observed that the soluble mediators produced by non-infected or Poly (I:C) transfected fibroblasts induced allogenic T cell proliferation, but mediators produced by DENV-2 infected fibroblasts inhibited this phenomenon. Additionally, the effects of fibroblast soluble mediators on CD14+ monocytes were analyzed to assess whether they affected the differentiation of monocyte derived dendritic cells (moDC). Our data showed that mediators produced by infected fibroblasts induced variable levels of monocyte differentiation into dendritic cells, even in the presence of recombinant GM-CSF and IL-4. Cells with dendritic cell-like morphology appeared in the culture; however, flow cytometry analysis showed that the mediators did not fully downregulate CD14 nor did they upregulate CD1a. Our data revealed that fibroblast-dendritic cell crosstalk promoted an antiviral response mediated manly by type I interferons over fibroblasts. Furthermore, the maturation of dendritic cells and T cell proliferation were promoted, which was inhibited by DENV-2-induced mediators. Together, our results suggest that activation of the adaptive immune response is influenced by the crosstalk of skin resident cells and the intensity of innate immune responses established in the microenvironment of the infected skin

Glycosylation and Serological Reactivity of an Expression-enhanced SARS-CoV-2 Viral Spike Mimetic

Extensive glycosylation of viral glycoproteins is a key feature of the antigenic surface of viruses and yet glycan processing can also be influenced by the manner of their recombinant production. The low yields of the soluble form of the trimeric spike (S) glycoprotein from SARS-CoV-2 has prompted advances in protein engineering that have greatly enhanced the stability and yields of the glycoprotein. The latest expression-enhanced version of the spike incorporates six proline substitutions to stabilize the prefusion conformation (termed SARS-CoV-2 S HexaPro). Although the substitutions greatly enhanced expression whilst not compromising protein structure, the influence of these substitutions on glycan processing has not been explored. Here, we show that the site-specific N-linked glycosylation of the expression-enhanced HexaPro resembles that of an earlier version containing two proline substitutions (2P), and that both capture features of native viral glycosylation. However, there are site-specific differences in glycosylation of HexaPro when compared to 2P. Despite these discrepancies, analysis of the serological reactivity of clinical samples from infected individuals confirmed that both HexaPro and 2P protein are equally able to detect IgG, IgA, and IgM responses in all sera analysed. Moreover, we extend this observation to include an analysis of glycan engineered S protein, whereby all N-linked glycans were converted to oligomannose-type and conclude that serological activity is not impacted by large scale changes in glycosylation. These observations suggest that variations in glycan processing will not impact the serological assessments currently being performed across the globe.</p

Living legends: Adapting centuries-old Lakota stories for use in contemporary educational settings

This paper details how our immersion program has edited and tailored transcriptions of traditional Lakota stories from the pre-reservation era for use as learning materials by new generations of Lakota learners. Furthermore, this can be a model for such adaptation in other language regions