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. 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. 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 < 0.05). The predominant serotypes were 19F, 11, 6A/B/C/D and 10A. More than 50% of the pneumococcal isolates belonged to a serotype/serogroup not included in PCV13. Eighty per cent of the isolates were not susceptible to benzylpenicillin and non-susceptibility to ampicillin and ceftriaxone was also high (42 and 37% respectively). Almost all the isolates (94%) were resistant to trimethoprim-sulphamethoxazole, while 43% of the strains were resistant to ≥3 antibiotics. Our study shows alarmingly high levels of reduced susceptibility to commonly used antibiotics in pneumococci carried by healthy Congolese children. This highlights the importance of local antibiotic resistance surveillance and indicates the needs for the more appropriate use of antibiotics in the area. The results further indicate that improved living conditions are needed to reduce the pneumococcal burden, in addition to PCV13 vaccination.

A recombinant subunit vaccine (Shingrix^®) 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^®). 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^®, which is produced in fibroblasts, the recombinant gE of Shingrix^® 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^®. Firstly, recombinant gE produced in CHO cells (“Shingrix situation”) is more scarcely decorated by O-linked glycans than gE from human fibroblasts (“Zostavax situation”), 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^®, 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-inactivatedS. 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.

The role of glycoprotein G (gG-2) of herpes simplex virus type 2 (HSV-2) in viral pathogenesis remains poorly understood. gG-2 is cleaved into a secreted form (sgG-2) and a membrane-associated form (mgG-2), but the in vivo function of mgG-2 and the contribution of its glycosylation to immune responses have not been defined. Here, we provide a comprehensive characterization of the N- and O-linked glycosylation profile of mgG-2 and investigate its functional relevance for viral spread and vaccine-induced immunity. Using a mouse genital infection model, we show that an mgG-2-deficient HSV-2 mutant replicates in vaginal epithelial cells but is severely impaired in dissemination to dorsal root ganglia and the central nervous system, identifying mgG-2 as a key determinant of neuronal spread in vivo. In parallel, immunization with recombinant mgG-2 elicited strong humoral and Th1-polarized CD4 + T-cell responses and conferred protection against genital HSV-2 challenge. Importantly, glycosylation of mgG-2 was required for optimal immunogenicity and protection, as deglycosylated variants induced reduced CD4 + T-cell responses and allowed increased viral spread to neuronal tissues. Mechanistically, our findings suggest that glycosylation of mgG-2 modulates antigen recognition and shapes adaptive immune responses that limit viral dissemination after vaccination. Together, these results demonstrate that mgG-2 plays a critical role in HSV-2 pathogenesis and provide a strong rationale for targeting glycosylated mgG-2 in the development of both prophylactic and therapeutic vaccines against HSV-2.