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The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a conserved domain and a target for neutralizing antibodies. We defined the carbohydrate content of the recombinant RBD produced in different mammalian cells. We found a higher degree of complex-type N-linked glycans, with less sialylation and more fucosylation, when the RBD was produced in human embryonic kidney cells compared to the same protein produced in Chinese hamster ovary cells. The carbohydrates on the RBD proteins were enzymatically modulated, and the effect on antibody reactivity was evaluated with serum samples from SARS-CoV-2 positive patients. Removal of all carbohydrates diminished antibody reactivity, while removal of only sialic acids or terminal fucoses improved the reactivity. The RBD produced in Lec3.2.8.1-cells, which generate carbohydrate structures devoid of sialic acids and with reduced fucose content, exhibited enhanced antibody reactivity, verifying the importance of these specific monosaccharides. The results can be of importance for the design of future vaccine candidates, indicating that it is possible to enhance the immunogenicity of recombinant viral proteins.

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is a conserved domain and a target for neutralizing antibodies. We defined the carbohydrate content of recombinant RBD produced in different mammalian cells. We found a higher degree of complex type N-linked glycans, with less sialylation and more fucosylation, when the RBD was produced in Human embryonic kidney cells compared to the same protein produced in Chinese hamster ovary cells. The carbohydrates on the RBD proteins were enzymatically modulated and the effect on antibody reactivity was evaluated with serum samples from SARS-CoV-2 positive patients. Removal of all carbohydrates diminished antibody reactivity while removal of only sialic acids or terminal fucoses improved the reactivity. The RBD produced in Lec3.2.8.1-cells, which generate carbohydrate structures devoid of sialic acids and with reduced fucose content, exhibited enhanced antibody reactivity verifying the importance of these specific monosaccharides. The results can be of importance for the design of future vaccine candidates, indicating that it might be possible to enhance the immunogenicity of recombinant viral proteins.

Background Pneumococcal conjugate vaccines have been introduced in the infant immunisation programmes in many countries to reduce the rate of fatal pneumococcal infections. In the Democratic Republic of the Congo (DR Congo) a 13-valent vaccine (PCV13) was introduced in 2013. Data on the burden of circulating pneumococci among children after this introduction are lacking. In this study, we aimed to determine the risk factors related to pneumococcal carriage in healthy Congolese children after the vaccine introduction and to assess the antibiotic resistance rates and serotype distribution among the isolated pneumococci. Methods In 2014 and 2015, 794 healthy children aged one to 60 months attending health centres in the eastern part of DR Congo for immunisation or growth monitoring were included in the study. Data on socio-demographic and medical factors were collected by interviews with the children’s caregivers. Nasopharyngeal swabs were obtained from all the children for bacterial culture, and isolated pneumococci were further tested for antimicrobial resistance using disc diffusion tests and, when indicated, minimal inhibitory concentration (MIC) determination, and for serotype/serogroup by molecular testing. Results The pneumococcal detection rate was 21%, being higher among children who had not received PCV13 vaccination, lived in rural areas, had an enclosed kitchen, were malnourished or presented with fever (p value

Hepatitis E virus (HEV) can cause chronic infection and liver cirrhosis in immunocompromised individuals. The frequency and clinical importance of HEV was studied retrospectively in a cohort of 236 Swedish allogeneic hematopoietic stem cell transplantation (HSCT) recipients. In blood samples collected at 6 months after HSCT, HEV RNA was identified in 8/236 (3.4%) patients, and 11/236 (4.7%) patients had detectable anti-HEV IgG and/or IgM, eight of whom were HEV RNA negative. Two of the patients with positive HEV RNA died with ongoing signs of hepatitis: one of acute liver and multiple organ failure, the other of unrelated causes. The remaining six patients with HEV RNA had cleared the infection at 7-24 (median 8.5) months after HSCT. HEV infection was associated with elevated alanine aminotransferase at 6 months after HSCT (OR 15, 1.3-174, p = 0.03). Active graft-versus-host disease of the liver at 6 months after HSCT was present in 3/8 (38%) patients with HEV RNA, but was not significantly associated with HEV infection. In conclusion, HEV infection is an important differential diagnosis in patients with elevated liver enzymes after HSCT. Although spontaneous clearance was common, the clinical course may be severe.

A recombinant subunit vaccine (Shingrix&reg, ) was recently licensed for use against herpes zoster. This vaccine is based on glycoprotein E (gE) of varicella zoster virus (VZV), the most abundantly expressed protein of VZV, harboring sites for N- and O-linked glycosylation. The subunit vaccine elicits stronger virus-specific CD4+ T cell response as well as antibody B cell response to gE, compared to the currently used live attenuated vaccine (Zostavax&reg, ). This situation is at variance with the current notion since a live vaccine, causing an active virus infection, should be far more efficient than a subunit vaccine based on only one single viral glycoprotein. We previously found gE to be heavily glycosylated, not least by numerous clustered O-linked glycans, when it was produced in human fibroblasts. However, in contrast to Zostavax&reg, which is produced in fibroblasts, the recombinant gE of Shingrix&reg, is expressed in Chinese hamster ovary (CHO) cells. Hence, the glycan occupancy and glycan structures of gE may differ considerably between the two vaccine types. Here, we aimed at (i) defining the glycan structures and positions of recombinant gE and (ii) identifying possible features of the recombinant gE O-glycosylation pattern contributing to the vaccine efficacy of Shingrix&reg, Firstly, recombinant gE produced in CHO cells (&ldquo, Shingrix situation&rdquo, ) is more scarcely decorated by O-linked glycans than gE from human fibroblasts (&ldquo, Zostavax situation&rdquo, ), with respect to glycan site occupancy. Secondly, screening of immunodominant B cell epitopes of gE, using a synthetic peptide library against serum samples from VZV-seropositive individuals, revealed that the O-linked glycan signature promoted binding of IgG antibodies via a decreased number of interfering O-linked glycans, but also via specific O-linked glycans enhancing antibody binding. These findings may, in part, explain the higher protective efficacy of Shingrix&reg, and can also be of relevance for development of subunit vaccines to other enveloped viruses.

A peculiar trait of pneumococci ( Streptococcus pneumoniae ) is their propensity to undergo spontaneous lysis during stationary growth due to activation of the enzyme autolysin (LytA), which fragments the peptidoglycan cell wall. The fragments that are generated upon autolysis impair phagocytosis and reduce production of interleukin-12 (IL-12) and gamma interferon (IFN-γ) by human leukocytes in response to intact pneumococci, thereby impeding crucial host defenses. The objective was to identify additional monocyte genes whose transcription is induced by intact pneumococci and subverted by autolyzed bacteria. Monocytes were isolated from healthy blood donors and stimulated for 3 h with UV-inactivated S. pneumoniae (Rx1PLY − LytA + strain), which is capable of autolyzing, its LytA − isogenic autolysin-deficient mutant, or a mixture of the two (containing twice the initial bacterial concentration). Gene expression was assessed by Illumina microarray, and selected findings were confirmed by reverse transcription-quantitative real-time PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and flow cytometry. In all, we identified 121 genes that were upregulated to a significantly higher degree by intact than autolyzed pneumococci. These included IFNB1 and a large set of interferon-induced genes, such as IFIT3, RSAD2, CFCL1, and CXCL10 genes, as well as IL12B and CD40 genes. RT-qPCR revealed that transcription of these genes in response to intact pneumococci diminished when autolyzed pneumococci were admixed and that this pattern was independent of pneumolysin. Thus, transcription of interferon-related genes is triggered by intact pneumococci and subverted by fragments generated by spontaneous bacterial autolysis. We suggest that interferon-related pathways are important for elimination of pneumococci and that autolysis contributes to virulence by extinguishing these pathways.

Herpes simplex virus type 1 has the ability to induce expression of a human gene cluster located on chromosome 19 upon infection. This gene cluster contains three fucosyltransferases (encoded by FUT3, FUT5 and FUT6) with the ability to add a fucose to an N-acetylglucosamine residue. Little is known regarding the transcriptional activation of these three genes in human cells. Intriguingly, herpes simplex virus type 1 activates all three genes simultaneously during infection, a situation not observed in uninfected tissue, pointing towards a virus specific mechanism for transcriptional activation. The aim of this study was to define the underlying mechanism for the herpes simplex virus type 1 activation of FUT3, FUT5 and FUT6 transcription. The transcriptional activation of the FUT-gene cluster on chromosome 19 in fibroblasts was specific, not involving adjacent genes. Moreover, inhibition of NFκB signaling through panepoxydone treatment significantly decreased the induction of FUT3, FUT5 and FUT6 transcriptional activation, as did siRNA targeting of p65, in herpes simplex virus type 1 infected fibroblasts. NFκB and p65 signaling appears to play an important role in the regulation of FUT3, FUT5 and FUT6 transcriptional activation by herpes simplex virus type 1 although additional, unidentified, viral factors might account for part of the mechanism as direct interferon mediated stimulation of NFκB was not sufficient to induce the fucosyltransferase encoding gene cluster in uninfected cells.