Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

Background: Chikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively. Results: Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits. Conclusions: Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections

Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

Background: Chikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively. Results: Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits. Conclusions: Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections

Earth observation for sustainable urban planning in developing countries: needs, trends, and future directions

Abstract: Cities are constantly changing and authorities face immense challenges in obtaining accurate and timely data to effectively manage urban areas. This is particularly problematic in the developing world where municipal records are often unavailable or not updated. Spaceborne earth observation (EO) has great potential for providing up-to-date spatial information about urban areas. This article reviews the application of EO for supporting urban planning. In particular, the article overviews case studies where EO was used to derive products and indicators required by urban planners. The review concludes that EO has sufficiently matured in recent years but that a shift from the current focus on purely science-driven EO applications to the provision of useful information for day-to-day decision-making and urban sustainability monitoring is clearly needed

Cross-Reactive T Cells Are Involved in Rapid Clearance of 2009 Pandemic H1N1 Influenza Virus in Nonhuman Primates

In mouse models of influenza, T cells can confer broad protection against multiple viral subtypes when antibodies raised against a single subtype fail to do so. However, the role of T cells in protecting humans against influenza remains unclear. Here we employ a translational nonhuman primate model to show that cross-reactive T cell responses play an important role in early clearance of infection with 2009 pandemic H1N1 influenza virus (H1N1pdm). To “prime” cellular immunity, we first infected 5 rhesus macaques with a seasonal human H1N1 isolate. These animals made detectable cellular and antibody responses against the seasonal H1N1 isolate but had no neutralizing antibodies against H1N1pdm. Four months later, we challenged the 5 “primed” animals and 7 naive controls with H1N1pdm. In naive animals, CD8+ T cells with an activated phenotype (Ki-67+ CD38+) appeared in blood and lung 5–7 days post inoculation (p.i.) with H1N1pdm and reached peak magnitude 7–10 days p.i. In contrast, activated T cells were recruited to the lung as early as 2 days p.i. in “primed” animals, and reached peak frequencies in blood and lung 4–7 days p.i. Interferon (IFN)-γ Elispot and intracellular cytokine staining assays showed that the virus-specific response peaked earlier and reached a higher magnitude in “primed” animals than in naive animals. This response involved both CD4+ and CD8+ T cells. Strikingly, “primed” animals cleared H1N1pdm infection significantly earlier from the upper and lower respiratory tract than the naive animals did, and before the appearance of H1N1pdm-specific neutralizing antibodies. Together, our results suggest that cross-reactive T cell responses can mediate early clearance of an antigenically novel influenza virus in primates. Vaccines capable of inducing such cross-reactive T cells may help protect humans against severe disease caused by newly emerging pandemic influenza viruses