Herpesviruses are among the most complex and widespread viruses, infection and propagation of which depend on envelope proteins. These proteins serve as mediators of cell entry as well as modulators of the immune response and are attractive vaccine targets. Although envelope proteins are known to carry glycans, little is known about the distribution, nature, and functions of these modifications. This is particularly true for O-glycans, thus we have recently developed a "bottom-up" mass spectrometry-based technique for mapping O-glycosylation sites on herpes simplex virus type 1 (HSV-1). We found wide distribution of O-glycans on HSV-1 glycoproteins and demonstrated that elongated O-glycans were essential for HSV-1 propagation. Here, we applied our proteome-wide discovery platform for mapping O-glycosites on representative and clinically significant members of the herpesvirus family -- varicella zoster virus (VZV), human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV). We identified a large number of O-glycosites distributed on most envelope proteins in all viruses and further demonstrated conserved patterns of O-glycans on distinct homologous proteins. Since glycosylation is highly dependent on the host cell, we tested VZV-infected cell lysates and clinically isolated virus and found evidence of consistent O-glycosites. These results present a comprehensive view of herpesvirus O-glycosylation and point to the widespread occurrence of O-glycans in regions of envelope proteins important for virus entry, formation, and recognition by the host's immune system. This knowledge enables dissection of specific functional roles of individual glycosites, and, moreover, provides a framework for design of glycoprotein vaccines with representative glycosylation.

Glycosylation of viral envelope proteins is important for infectivity and interaction with host immunity, however, our current knowledge of the functions of glycosylation is largely limited to N-glycosylation because it is difficult to predict and identify site-specific O-glycosylation. Here, we present a novel proteome-wide discovery strategy for O-glycosylation sites on viral envelope proteins using herpes simplex virus type 1 (HSV-1) as a model. We identified 74 O-linked glycosylation sites on 8 out of the 12 HSV-1 envelope proteins. Two of the identified glycosites found in glycoprotein B were previously implicated in virus attachment to immune cells. We show that HSV-1 infection distorts the secretory pathway and that infected cells accumulate glycoproteins with truncated O-glycans, nonetheless retaining the ability to elongate most of the surface glycans. With the use of precise gene editing, we further demonstrate that elongated O-glycans are essential for HSV-1 in human HaCaT keratinocytes, where HSV-1 produced markedly lower viral titers in HaCaT with abrogated O-glycans compared to the isogenic counterpart with normal O-glycans. The roles of O-linked glycosylation for viral entry, formation, secretion, and immune recognition are poorly understood, and the O-glycoproteomics strategy presented here now opens for unbiased discovery on all enveloped viruses. Information on site-specific O-glycosylation of viral envelope glycoproteins is generally very limited despite important functions. We present a powerful mass-spectrometry based strategy to globally identify O-glycosylation sites on viral envelope proteins of a given virus in the context of a productive infection. We successfully utilized the strategy to map O-linked glycosylation sites on the complex HSV-1 virus demonstrating that O-glycosylation is widely distributed on most envelope proteins. Moreover, we used genetically engineered keratinocytes lacking O-glycan elongation capacity to demonstrate that O-linked glycans are indeed important for HSV-1 biology as HSV-1 particles produced in these cells had significantly lower titers compared to wild-type keratinocytes. These tools enable wider discovery and detailed analysis of the role of site-specific O-glycosylation in virology.

Background: Major hurdles for survival after lung transplantation are rejections and infectious complications. Adequate methods for monitoring immune suppression status are lacking. Here, we evaluated quantification of torque teno virus (TTV) and Epstein-Barr virus (EBV) as biomarkers for defining the net state of immunosuppression in lung-transplanted patients.Methods: This prospective single-center study included 98 patients followed for 2 years after transplantation. Bacterial infections, fungal infections, viral respiratory infections (VRTI), cytomegalovirus (CMV) viremia, and acute rejections, as well as TTV and EBV levels, were monitored.Results: The levels of torque teno virus DNA increased rapidly after transplantation, likely due to immunosuppressive treatment. A modest increase in levels of Epstein-Barr virus DNA was also observed after transplantation. There were no associations between either TTV or EBV and infectious events or acute rejection, respectively, during follow-up. When Tacrolimus was the main immunosuppressive treatment, TTV DNA levels were significantly elevated 6–24 months after transplantation as compared with Cyclosporine treatment.Conclusions: Although replication of TTV, but not EBV, appears to reflect the functionality of the immune system, depending on the type of immunosuppressive treatment, quantification of TTV or EBV as biomarkers has limited potential for defining the net state of immune suppression.

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.

Background: Chronic lung allograft dysfunction (CLAD) is the major limiting factor for long-term survival in lung transplant recipients. Viral respiratory tract infection (VRTI) has been previously associated with CLAD development. The main purpose of this study was to evaluate the long-term effects of VRTI during the first year after lung transplantation in relation to CLAD development. Method: Ninety-eight patients undergoing lung transplantation were prospectively enrolled between 2009 and 2012. They were monitored for infections with predefined intervals and on extra visits during the first year, the total follow-up period ranged between 5 and 8 years. Nasopharyngeal swab and bronchoalveolar lavage samples were analyzed using a multiplex polymerase chain reaction panel for respiratory pathogens. Data regarding clinical characteristics and infectious events were recorded. Results: Viral respiratory tract infection during the first year was identified as a risk factor for long-term CLAD development (P = 0.041, hazard ratio 1.94 [1.03-3.66]) in a time-dependent multivariate Cox regression analysis. We also found that coronavirus in particular was associated with increased risk for CLAD development. Other identified risk factors were acute rejection and cyclosporine treatment. Conclusions: This study suggests that VRTI during the first year after lung transplantation is associated with long-term CLAD development and that coronavirus infections in particular might be a risk factor.

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.