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The ability of DENV2 to display different morphologies (hence different antigenic properties) complicates vaccine and therapeutics development. Previous studies showed most strains of laboratory adapted DENV2 particles changed from smooth to “bumpy” surfaced morphology when the temperature is switched from 29°C at 37°C. Here we identified five envelope (E) protein residues different between two alternative passage history DENV2 NGC strains exhibiting smooth or bumpy surface morphologies. Several mutations performed on the smooth DENV2 infectious clone destabilized the surface, as observed by cryoEM. Molecular dynamics simulations demonstrated how chemically subtle substitution at various positions destabilized dimeric interactions between E proteins. In contrast, three out of four DENV2 clinical isolates showed a smooth surface morphology at 37°C, and only at high fever temperature (40°C) did they become “bumpy”. These results imply vaccines should contain particles representing both morphologies. For prophylactic and therapeutic treatments, this study also informs on which types of antibodies should be used at different stages of an infection, i.e., those that bind to monomeric E proteins on the bumpy surface or across multiple E proteins on the smooth surfaced virus. [ABSTRACT FROM AUTHOR]

The formation and release of outer membrane vesicles (OMVs) is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila), a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a’s targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host. [ABSTRACT FROM AUTHOR]

Salmonella enterica infections are a significant global health issue, and development of vaccines against these bacteria requires an improved understanding of how vaccination affects the growth and spread of the bacteria within the host. We have combined in vivo tracking of molecularly tagged bacterial subpopulations with mathematical modelling to gain a novel insight into how different classes of vaccines and branches of the immune response protect against secondary Salmonella enterica infections of the mouse. We have found that a live Salmonella vaccine significantly reduced bacteraemia during a secondary challenge and restrained inter-organ spread of the bacteria in the systemic organs. Further, fitting mechanistic models to the data indicated that live vaccine immunisation enhanced both the bacterial killing in the very early stages of the infection and bacteriostatic control over the first day post-challenge. T-cell immunity induced by this vaccine is not necessary for the enhanced bacteriostasis but is required for subsequent bactericidal clearance of Salmonella in the blood and tissues. Conversely, a non-living vaccine while able to enhance initial blood clearance and killing of virulent secondary challenge bacteria, was unable to alter the subsequent bacterial growth rate in the systemic organs, did not prevent the resurgence of extensive bacteraemia and failed to control the spread of the bacteria in the body. [ABSTRACT FROM AUTHOR]