Interactions between High Seroreactivity to Human Herpes Virus 6A and Epstein-Barr Virus in MS Development: A Presymptomatic Case-Control Study.

Synergistic interactions between human herpesvirus 6A (HHV-6A) and Epstein-Barr virus (EBV) are hypothesized in the etiopathogenesis of multiple sclerosis (MS). This study investigated if HHV-6A and EBV seroreactivities interact regarding the risk of developing MS. Antibodies against viral antigens were analyzed in biobank samples from 670 individuals who later developed MS and matched controls. Additive interactions were analyzed. A significant interaction between HHV-6A and EBNA-1 seroreactivities was observed in study participants above the median age of 24.9 years (attributable proportion due to interaction = 0.45). This finding supports the hypothesis that HHV-6A and EBV infections interact in MS development. ANN NEUROL 2024.

Human herpesvirus 6A and axonal injury before the clinical onset of multiple sclerosis.

Recent research indicates that multiple sclerosis is preceded by a prodromal phase with elevated levels of serum neurofilament light chain (sNfL), a marker of axonal injury. The effect of environmental risk factors on the extent of axonal injury during this prodrome is unknown. Human herpesvirus 6A (HHV-6A) is associated with an increased risk of developing multiple sclerosis. The objective of this study was to determine if HHV-6A serostatus is associated with the level of sNfL in the multiple sclerosis prodrome, which would support a causative role of HHV-6A. A nested case-control study was performed by crosslinking multiple sclerosis registries with Swedish biobanks. Individuals with biobank samples collected before the clinical onset of multiple sclerosis were included as cases. Controls without multiple sclerosis were randomly selected, matched for biobank, sex, sampling date and age. Serostatus of HHV-6A and Epstein-Barr virus was analysed with a bead-based multiplex assay. The concentration of sNfL was analysed with single molecule array technology. The association between HHV-6A serology and sNfL was assessed by stratified t-tests and linear regressions, adjusted for Epstein-Barr virus serostatus and sampling age. Within-pair ratios of HHV-6A seroreactivity and sNfL were calculated for each case and its matched control. To assess the temporal relationship between HHV-6A antibodies and sNfL, these ratios were plotted against the time to the clinical onset of multiple sclerosis and compared using locally estimated scatterplot smoothing regressions with 95% confidence intervals (CI). Samples from 519 matched case-control pairs were included. In cases, seropositivity of HHV-6A was significantly associated with the level of sNfL (+11%, 95% CI 0.2-24%, P = 0.045) and most pronounced in the younger half of the cases (+24%, 95% CI 6-45%, P = 0.007). No such associations were observed among the controls. Increasing seroreactivity against HHV-6A was detectable before the rise of sNfL (significant within-pair ratios from 13.6 years versus 6.6 years before the clinical onset of multiple sclerosis). In this study, we describe the association between HHV-6A antibodies and the degree of axonal injury in the multiple sclerosis prodrome. The findings indicate that elevated HHV-6A antibodies both precede and are associated with a higher degree of axonal injury, supporting the hypothesis that HHV-6A infection may contribute to multiple sclerosis development in a proportion of cases.

Identification of epidermal growth factor receptor-tyrosine kinase inhibitor targeting the VP1 pocket of human rhinovirus.

Screening a library of 1,200 preselected kinase inhibitors for anti-human rhinovirus 2 (HRV-2) activity in HeLa cells identified a class of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) as effective virus blockers. These were based on the 4-anilinoquinazoline-7-oxypiperidine scaffold, with the most potent representative AZ5385 inhibiting the virus with EC50 of 0.35 µM. Several structurally related analogs confirmed activity in the low µM range, while interestingly, other TKIs targeting EGFR lacked anti-HRV-2 activity. To further probe this lack of association between antiviral activity and EGFR inhibition, we stained infected cells with antibodies specific for activated EGFR (Y1068) and did not observe a dependency on EGFR-TK activity. Instead, consecutive passages of HRV-2 in HeLa cells in the presence of a compound and subsequent nucleotide sequence analysis of resistant viral variants identified the S181T and T210A alterations in the major capsid VP1 protein, with both residues located in the vicinity of a known hydrophobic pocket on the viral capsid. Further characterization of the antiviral effects of AZ5385 showed a modest virus-inactivating (virucidal) activity, while anti-HRV-2 activity was still evident when the inhibitor was added as late as 10 h post infection. The RNA copy/infectivity ratio of HRV-2 propagated in AZ5385 presence was substantially higher than that of control HRV indicating that the compound preferentially targeted HRV progeny virions during their maturation in infected cells. Besides HRV, the compound showed anti-respiratory syncytial virus activity, which warrants its further studies as a candidate compound against viral respiratory infections.

Seroreactivity against lytic, latent and possible cross-reactive EBV antigens appears on average 10 years before MS induced preclinical neuroaxonal damage.

BACKGROUND: Multiple sclerosis (MS) and presymptomatic axonal injury appear to develop only after an Epstein-Barr virus (EBV) infection. This association remains to be confirmed across a broad preclinical time range, for lytic and latent EBV seroreactivity, and for potential cross-reacting antigens. METHODS: We performed a case-control study with 669 individual serum samples obtained before clinical MS onset, identified through cross-linkage with the Swedish MS register. We assayed antibodies against EBV nuclear antigen 1 (EBNA1), viral capsid antigen p18, glycoprotein 350 (gp350), the potential cross-reacting protein anoctamin 2 (ANO2) and the level of sNfL, a marker of axonal injury. RESULTS: EBNA1 (latency) seroreactivity increased in the pre-MS group, at 15-20 years before clinical MS onset, followed by gp350 (lytic) seroreactivity (p=0.001-0.009), ANO2 seropositivity appeared shortly after EBNA1-seropositivity in 16.7% of pre-MS cases and 10.0% of controls (p=0.001).With an average lag of almost a decade after EBV, sNfL gradually increased, mainly in the increasing subgroup of seropositive pre-MS cases (p=8.10-5 compared with non-MS controls). Seropositive pre-MS cases reached higher sNfL levels than seronegative pre-MS (p=0.038). In the EBNA1-seropositive pre-MS group, ANO2 seropositive cases had 26% higher sNfL level (p=0.0026). CONCLUSIONS: Seroreactivity against latent and lytic EBV antigens, and in a subset ANO2, was detectable on average a decade before the appearance of a gradually increasing axonal injury occurring in the last decade before the onset of clinical MS. These findings strengthen the hypothesis of latent EBV involvement in the pathogenesis of MS.

Antiviral Rotenoids and Isoflavones Isolated from Millettia oblata ssp. teitensis.

Three new (1-3) and six known rotenoids (5-10), along with three known isoflavones (11-13), were isolated from the leaves of Millettia oblata ssp. teitensis. A new glycosylated isoflavone (4), four known isoflavones (14-18), and one known chalcone (19) were isolated from the root wood extract of the same plant. The structures were elucidated by NMR and mass spectrometric analyses. The absolute configuration of the chiral compounds was established by a comparison of experimental ECD and VCD data with those calculated for the possible stereoisomers. This is the first report on the use of VCD to assign the absolute configuration of rotenoids. The crude leaves and root wood extracts displayed anti-RSV (human respiratory syncytial virus) activity with IC50 values of 0.7 and 3.4 µg/mL, respectively. Compounds 6, 8, 10, 11, and 14 showed anti-RSV activity with IC50 values of 0.4-10 µM, while compound 3 exhibited anti-HRV-2 (human rhinovirus 2) activity with an IC50 of 4.2 µM. Most of the compounds showed low cytotoxicity for laryngeal carcinoma (HEp-2) cells; however compounds 3, 11, and 14 exhibited low cytotoxicity also in primary lung fibroblasts. This is the first report on rotenoids showing antiviral activity against RSV and HRV viruses.

Seroprevalence of tick-borne encephalitis virus and vaccination coverage of tick-borne encephalitis, Sweden, 2018 to 2019.

BackgroundIn Sweden, information on seroprevalence of tick-borne encephalitis virus (TBEV) in the population, including vaccination coverage and infection, is scattered. This is largely due to the absence of a national tick-borne encephalitis (TBE) vaccination registry, scarcity of previous serological studies and use of serological methods not distinguishing between antibodies induced by vaccination and infection. Furthermore, the number of notified TBE cases in Sweden has continued to increase in recent years despite increased vaccination.AimThe aim was to estimate the TBEV seroprevalence in Sweden.MethodsIn 2018 and 2019, 2,700 serum samples from blood donors in nine Swedish regions were analysed using a serological method that can distinguish antibodies induced by vaccination from antibodies elicited by infection. The regions were chosen to reflect differences in notified TBE incidence.ResultsThe overall seroprevalence varied from 9.7% (95% confidence interval (CI): 6.6-13.6%) to 64.0% (95% CI: 58.3-69.4%) between regions. The proportion of vaccinated individuals ranged from 8.7% (95% CI: 5.8-12.6) to 57.0% (95% CI: 51.2-62.6) and of infected from 1.0% (95% CI: 0.2-3.0) to 7.0% (95% CI: 4.5-10.7). Thus, more than 160,000 and 1,600,000 individuals could have been infected by TBEV and vaccinated against TBE, respectively. The mean manifestation index was 3.1%.ConclusionA difference was observed between low- and high-incidence TBE regions, on the overall TBEV seroprevalence and when separated into vaccinated and infected individuals. The estimated incidence and manifestation index argue that a large proportion of TBEV infections are not diagnosed.

Serologic immunogenicity and safety of Herpes Zoster subunit vaccine in patients with Rheumatoid Arthritis receiving Janus Kinase inhibitors.

OBJECTIVE: Patients with rheumatoid arthritis (RA) treated with Janus Kinase inhibitors (JAKi) are at increased risk of Herpes Zoster (HZ). The objective of this study was to investigate serological immunogenicity and safety of the HZ subunit (HZ/su) vaccine in RA patients treated with JAKi, for which little is known. METHODS: RA patients treated with JAKi (n = 82) at the Department of Rheumatology, Skåne University Hospital, Sweden, and healthy controls (n = 51) received two doses of the HZ/su vaccine (Shingrix). Vaccine-specific antibody responses were analysed using indirect enzyme-linked immunosorbent assay (ELISA). Post-vaccination antibody levels were compared between patients and controls using analysis of covariance. Potential predictors for vaccine response were investigated using a multivariable linear regression analysis. Self-reported adverse events (AEs) and changes in RA disease activity were analysed. RESULTS: Following vaccination, vaccine-specific antibody levels increased significantly in both patients and controls (p< 0.0001). 80.5% of patients and 98.0% of controls achieved a ≥ 4-fold increase in antibody levels. Post-vaccination antibody levels were lower in patients than controls (ratio 0.44, 95% CI 0.31-0.63), and lower in patients receiving JAKi+Methotrexate than JAKi monotherapy (ratio 0.43, 95% CI 0.24-0.79). AEs, mostly mild/moderate, were common. One patient developed HZ and six patients (6.5%) had increased RA disease activity following vaccination. CONCLUSION: The HZ/su vaccine was serologically immunogenic in most RA patients treated with JAKi. Moreover, the vaccine had an acceptable safety profile. These results support recommendations for usage of the HZ/su vaccine in this vulnerable population. TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, NCT03886038.

Crotofolane Diterpenoids and Other Constituents Isolated from Croton kilwae.

Six new crotofolane diterpenoids (1-6) and 13 known compounds (7-19) were isolated from the MeOH-CH2Cl2 (1:1, v/v) extracts of the leaves and stem bark of Croton kilwae. The structures of the new compounds were elucidated by extensive analysis of spectroscopic and mass spectrometric data. The structure of crotokilwaepoxide A (1) was confirmed by single-crystal X-ray diffraction, allowing for the determination of its absolute configuration. The crude extracts and the isolated compounds were investigated for antiviral activity against respiratory syncytial virus (RSV) and human rhinovirus type-2 (HRV-2) in HEp-2 and HeLa cells, respectively, for antibacterial activity against the Gram-positive Bacillus subtilis and the Gram-negative Escherichia coli, and for antimalarial activity against the Plasmodium falciparum Dd2 strain. ent-3ß,19-Dihydroxykaur-16-ene (7) and ayanin (16) displayed anti-RSV activities with IC50 values of 10.2 and 6.1 µM, respectively, while exhibiting only modest cytotoxic effects on HEp-2 cells that resulted in selectivity indices of 4.9 and 16.4. Compounds 2 and 5 exhibited modest anti-HRV-2 activity (IC50 of 44.6 µM for both compounds), while compound 16 inhibited HRV-2 with an IC50 value of 1.8 µM. Compounds 1-3 showed promising antiplasmodial activities (80-100% inhibition) at a 50 µM concentration.

Occupational risks for infection with influenza A and B: a national case-control study covering 1 July 2006-31 December 2019.

OBJECTIVES: We investigated whether crowded workplaces, sharing surfaces and exposure to infections were factors associated with a positive test for influenza virus. METHODS: We studied 11 300 cases with a positive test for influenza A and 3671 cases of influenza B from Swedish registry of communicable diseases. Six controls for each case were selected from the population registry, with each control being assigned the index date of their corresponding case. We linked job histories to job-exposure matrices (JEMs), to assess different transmission dimensions of influenza and risks for different occupations compared with occupations that the JEM classifies as low exposed. We used adjusted conditional logistic analyses to estimate the ORs for influenza with 95% CI. RESULTS: The highest odds were for influenza were: regular contact with infected patients (OR 1.64, 95% CI 1.54 to 1.73); never maintained social distance (OR 1.51, 95% CI 1.43 to 1.59); frequently sharing materials/surfaces with the general public (OR 1.41, 95% CI 1.34 to 1.48); close physical proximity (OR 1.54, 95% CI 1.45 to 1.62) and high exposure to diseases or infections (OR 1.54, 95% CI 1.44 to 1.64). There were small differences between influenza A and influenza B. The five occupations with the highest odds as compared with low exposed occupations were: primary care physicians, protective service workers, elementary workers, medical and laboratory technicians, and taxi drivers. CONCLUSIONS: Contact with infected patients, low social distance and sharing surfaces are dimensions that increase risk for influenza A and B. Further safety measures are needed to diminish viral transmission in these contexts.

Modified ent-Abietane Diterpenoids from the Leaves of Suregada zanzibariensis.

The leaf extract of Suregada zanzibariensis gave two new modified ent-abietane diterpenoids, zanzibariolides A (1) and B (2), and two known triterpenoids, simiarenol (3) and ß-amyrin (4). The structures of the isolated compounds were elucidated based on NMR and MS data analysis. Single-crystal X-ray diffraction was used to establish the absolute configurations of compounds 1 and 2. The crude leaf extract inhibited the infectivity of herpes simplex virus 2 (HSV-2, IC50 11.5 µg/mL) and showed toxicity on African green monkey kidney (GMK AH1) cells at CC50 52 µg/mL. The isolated compounds 1-3 showed no anti-HSV-2 activity and exhibited insignificant toxicity against GMK AH1 cells at ≥100 µM.

An ABCA4 loss-of-function mutation causes a canine form of Stargardt disease.

Autosomal recessive retinal degenerative diseases cause visual impairment and blindness in both humans and dogs. Currently, no standard treatment is available, but pioneering gene therapy-based canine models have been instrumental for clinical trials in humans. To study a novel form of retinal degeneration in Labrador retriever dogs with clinical signs indicating cone and rod degeneration, we used whole-genome sequencing of an affected sib-pair and their unaffected parents. A frameshift insertion in the ATP binding cassette subfamily A member 4 (ABCA4) gene (c.4176insC), leading to a premature stop codon in exon 28 (p.F1393Lfs*1395), was identified. In contrast to unaffected dogs, no full-length ABCA4 protein was detected in the retina of an affected dog. The ABCA4 gene encodes a membrane transporter protein localized in the outer segments of rod and cone photoreceptors. In humans, the ABCA4 gene is associated with Stargardt disease (STGD), an autosomal recessive retinal degeneration leading to central visual impairment. A hallmark of STGD is the accumulation of lipofuscin deposits in the retinal pigment epithelium (RPE). The discovery of a canine homozygous ABCA4 loss-of-function mutation may advance the development of dog as a large animal model for human STGD.

Comparable endemic coronavirus nucleoprotein-specific antibodies in mild and severe Covid-19 patients.

The severity of disease of Covid-19 is highly variable, ranging from asymptomatic to critical respiratory disease and death. Potential cross-reactive immune responses between SARS-CoV-2 and endemic coronavirus (eCoV) may hypothetically contribute to this variability. We herein studied if eCoV nucleoprotein (N)-specific antibodies in the sera of patients with mild or severe Covid-19 are associated with Covid-19 severity. There were comparable levels of eCoV N-specific antibodies early and during the first month of infection in Covid-19 patients with mild and severe symptoms, and healthy SARS-CoV-2-negative subjects. These results warrant further studies to investigate the potential role of eCoV-specific antibodies in immunity to SARS-CoV-2 infection.

Cytomegalovirus seropositivity is associated with reduced risk of multiple sclerosis-a presymptomatic case-control study.

BACKGROUND AND PURPOSE: Epstein-Barr virus (EBV) and human herpesvirus 6A (HHV-6A) are associated with increased risk of multiple sclerosis (MS). Conversely, infection with cytomegalovirus (CMV) has been suggested to reduce the risk of MS but supporting data from presymptomatic studies are lacking. Here, it was sought to increase the understanding of CMV in MS aetiology. METHODS: A nested case-control study was performed with presymptomatically collected blood samples identified through crosslinkage of MS registries and Swedish biobanks. Serological antibody response against CMV, EBV and HHV-6A was determined using a bead-based multiplex assay. Odds ratio (OR) with 95% confidence interval (CI) for CMV seropositivity as a risk factor for MS was calculated by conditional logistic regression and adjusted for EBV and HHV-6A seropositivity. Potential interactions on the additive scale were analysed by calculating the attributable proportion due to interaction (AP). RESULTS: Serum samples from 670 pairs of matched cases and controls were included. CMV seropositivity was associated with a reduced risk for MS (OR = 0.70, 95% CI 0.56-0.88, p = 0.003). Statistical interactions on the additive scale were observed between seronegativity for CMV and seropositivity against HHV-6A (AP 0.34, 95% CI 0.06-0.61) and EBV antigen EBNA-1 (amino acid 385-420) at age 20-39 years (AP 0.37, 95% CI 0.09-0.65). CONCLUSIONS: Cytomegalovirus seropositivity is associated with a decreased risk for MS. The protective role for CMV infection in MS aetiology is further supported by the interactions between CMV seronegativity and EBV and HHV-6A seropositivity.

In Situ Assembly of Choline Acetyltransferase Ligands by a Hydrothiolation Reaction Reveals Key Determinants for Inhibitor Design.

The potential drug target choline acetyltransferase (ChAT) catalyses the production of the neurotransmitter acetylcholine in cholinergic neurons, T-cells, and B-cells. Herein, we show that arylvinylpyridiniums (AVPs), the most widely studied class of ChAT inhibitors, act as substrate in an unusual coenzyme A-dependent hydrothiolation reaction. This in situ synthesis yields an adduct that is the actual enzyme inhibitor. The adduct is deeply buried in the active site tunnel of ChAT and interactions with a hydrophobic pocket near the choline binding site have major implications for the molecular recognition of inhibitors. Our findings clarify the inhibition mechanism of AVPs, establish a drug modality that exploits a target-catalysed reaction between exogenous and endogenous precursors, and provide new directions for the development of ChAT inhibitors with improved potency and bioactivity.

Intrathecal immunoreactivity in people with or without previous infectious mononucleosis.

OBJECTIVES: The risk of developing multiple sclerosis (MS) increases (OR: 3.1) after infectious mononucleosis (IM). However, the nature of this link is obscure. We tested the hypothesis that IM might incur long-term sequelae, including low-key inflammatory activity, with characteristics of an MS endophenotype (or presymptomatic trait) and that assays of MS-relevant cyto-/chemokines in the cerebrospinal fluid (CSF) post-IM may show a trend in this direction. MATERIALS AND METHODS: We selected seven CSF cytokines (IL-1b, IL-6, YKL-40, TNF-alpha) or chemokines (IL-8, CCL2, IP-10), representing pro-inflammatory factors previously associated with MS. We assayed the CSF levels of these seven cyto-/chemokines in healthy individuals with a median follow-up time of 10 years after serologically confirmed IM (post-IM group, n = 22), and in healthy controls without a history of IM (n = 19). A group of MS patients (n = 23) were included as reference. RESULTS: The CSF levels of IP-10, YKL-40, and CCL-2 were higher in the post-IM group than in our IM unexposed controls (P = .021, .049, .028). Seven of seven cyto-/chemokine assays showed a trend in the predicted direction (P of binomial ratio = .008). However, this trend was non-significant in a multivariate test (P = .22). A power analysis indicated that similar studies including a larger cohort would be numerically realistic. CONCLUSIONS: These results do not reject the hypothesis that the established epidemiological association between IM and MS results from a stepwise inflammatory propagation from IM sequelae to an MS endophenotype (or presymptomatic trait) in a proportion of IM patients, pending confirmation with adequate power.

Correction to: Bacteria: back pain, leg pain and Modic sign-a surgical multicenter comparative study.

Unfortunately, the 5th author name was incorrectly published in the original paper. The complete correct name is given below.

Intrathecal complement activation by the classical pathway in tick-borne encephalitis.

Tick-borne encephalitis (TBE) is one of the most prevalent viral central nervous system (CNS) infections in Eurasia and neurological sequelae are common. The immune responses are considered crucial for the pathogenesis. The aim of this study was to explore the activation of the complement system in TBE. The complement system is a part of the innate immune response in the CNS, which previously has been reported to be activated in other flavivirus infections. We analyzed complement factors in 44 paired cerebrospinal fluid (CSF) and serum samples from 20 cases of TBE in the acute and later stages, as well as in serum and CSF from 32 healthy controls. The concentrations of complement factors C1q, C3a, C3b, and C5a were determined with commercially available ELISA kits. Clinical data to categorize the severity of disease and outcome was retrieved from the medical records of the TBE patients. We found significantly higher concentrations of all of the analyzed complement factors in the CSF from TBE patients compared to the healthy controls. In particular, the marked increment of C1q concentrations in the CSF (p < 0,001 as compared to controls) indicated an intrathecal activation by the classical pathway. There was no correlation between complement factor concentrations in the CSF and severity of the disease in the acute phase or with sequelae at 6 months follow-up. We have found an intrathecal complement activation in TBE, and the marked increase of complement factor C1q indicated an activation by the classical pathway.

Bacteria: back pain, leg pain and Modic sign-a surgical multicentre comparative study.

PURPOSE: To compare bacterial findings in pain-generating degenerated discs in adults operated on for lumbar disc herniation (LDH), and mostly also suffering from low back pain (LBP), with findings in adolescent patients with non-degenerated non-pain-generating discs operated on for scoliosis, and to evaluate associations with Modic signs on magnetic resonance imaging (MRI). Cutibacterium acnes (Propionibacterium acnes) has been found in painful degenerated discs, why it has been suggested treating patients with LDH/LBP with antibiotics. As multidrug-resistant bacteria are a worldwide concern, new indications for using antibiotics should be based on solid scientific evidence. METHODS: Between 2015 and 2017, 40 adults with LDH/LBP (median age 43, IQR 33-49) and 20 control patients with scoliosis (median age 17, IQR 15-20) underwent surgery at seven Swedish hospitals. Samples were cultured from skin, surgical wound, discs and vertebrae. Genetic relatedness of C. acnes isolates was investigated using single-nucleotide polymorphism analysis. DNA samples collected from discs/vertebrae were analysed using 16S rRNA-based PCR sequencing. MRI findings were assessed for Modic changes. RESULTS: No bacterial growth was found in 6/40 (15%) LDH patients, compared with 3/20 (15%) scoliosis patients. Most positive samples in both groups were isolated from the skin and then from subcutis or deep within the wound. Of the four disc and vertebral samples from each of the 60 patients, 235/240 (98%) were DNA negative by bacterial PCR. A single species, C. acnes, was found exclusively in the disc/vertebra from one patient in each group. In the LDH group, 29/40 (72%) patients had at least one sample with growth of C. acnes, compared to 14/20 (70%) in the scoliosis group. Bacterial findings and Modic changes were not associated. CONCLUSIONS: Cutibacterium acnes found in discs and vertebrae during surgery for disc herniation in adults with degenerated discs may be caused by contamination, as findings in this group were similar to findings in a control group of young patients with scoliosis and non-degenerated discs. Furthermore, such findings were almost always combined with bacterial findings on the skin and/or in the wound. There was no association between preoperative Modic changes and bacterial findings. Antibiotic treatment of lumbar disc herniation with sciatica and/or low back pain, without signs of clinical discitis/spondylitis, should be seriously questioned. These slides can be retrieved under Electronic Supplementary Material.

Measles outbreak in Gothenburg urban area, Sweden, 2017 to 2018: low viral load in breakthrough infections.

In an outbreak of measles in Gothenburg, Sweden, breakthrough infections (i.e. infections in individuals with a history of vaccination) were common. The objective of this study was to compare measles RNA levels between naïve (i.e. primary) and breakthrough infections. We also propose a fast provisional classification of breakthrough infections. Medical records were reviewed and real-time PCR-positive samples genotyped. Cases were classified as naïve, breakthrough or vaccine infections. We compared clinical symptoms and measles RNA cycle threshold (Ct) values between breakthrough and naïve infections. Sixteen of 28 confirmed cases of measles in this outbreak were breakthrough infections. A fast provisional classification, based on previous history of measles vaccination and detectable levels of measles IgG in acute serum, correctly identified 14 of the 16 breakthrough infections, confirmed by IgG avidity testing. Measles viral load was significantly lower in nasopharyngeal samples from individuals with breakthrough compared with naïve infections (median Ct-values: 32 and 19, respectively, p < 0.0001). No onward transmission from breakthrough infections was identified. Our results indicate that a high risk of onward transmission is limited to naïve infections. We propose a fast provisional classification of breakthrough measles that can guide contact tracing in outbreak settings.

Recombinant Glycoprotein E of Varicella Zoster Virus Contains Glycan-Peptide Motifs That Modulate B Cell Epitopes into Discrete Immunological Signatures.

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.