Efficacy, safety, and immunogenicity of a booster regimen of Ad26.COV2.S vaccine against COVID-19 (ENSEMBLE2): results of a randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Despite the availability of effective vaccines against COVID-19, booster vaccinations are needed to maintain vaccine-induced protection against variant strains and breakthrough infections. This study aimed to investigate the efficacy, safety, and immunogenicity of the Ad26.COV2.S vaccine (Janssen) as primary vaccination plus a booster dose. METHODS: ENSEMBLE2 is a randomised, double-blind, placebo-controlled, phase 3 trial including crossover vaccination after emergency authorisation of COVID-19 vaccines. Adults aged at least 18 years without previous COVID-19 vaccination at public and private medical practices and hospitals in Belgium, Brazil, Colombia, France, Germany, the Philippines, South Africa, Spain, the UK, and the USA were randomly assigned 1:1 via a computer algorithm to receive intramuscularly administered Ad26.COV2.S as a primary dose plus a booster dose at 2 months or two placebo injections 2 months apart. The primary endpoint was vaccine efficacy against the first occurrence of molecularly confirmed moderate to severe-critical COVID-19 with onset at least 14 days after booster vaccination, which was assessed in participants who received two doses of vaccine or placebo, were negative for SARS-CoV-2 by PCR at baseline and on serology at baseline and day 71, had no major protocol deviations, and were at risk of COVID-19 (ie, had no PCR-positive result or discontinued the study before day 71). Safety was assessed in all participants; reactogenicity, in terms of solicited local and systemic adverse events, was assessed as a secondary endpoint in a safety subset (approximately 6000 randomly selected participants). The trial is registered with ClinicalTrials.gov, NCT04614948, and is ongoing. FINDINGS: Enrolment began on Nov 16, 2020, and the primary analysis data cutoff was June 25, 2021. From 34 571 participants screened, the double-blind phase enrolled 31 300 participants, 14 492 of whom received two doses (7484 in the Ad26.COV2.S group and 7008 in the placebo group) and 11 639 of whom were eligible for inclusion in the assessment of the primary endpoint (6024 in the Ad26.COV2.S group and 5615 in the placebo group). The median (IQR) follow-up post-booster vaccination was 36·0 (15·0-62·0) days. Vaccine efficacy was 75·2% (adjusted 95% CI 54·6-87·3) against moderate to severe-critical COVID-19 (14 cases in the Ad26.COV2.S group and 52 cases in the placebo group). Most cases were due to the variants alpha (B.1.1.7) and mu (B.1.621); endpoints for the primary analysis accrued from Nov 16, 2020, to June 25, 2021, before the global dominance of delta (B.1.617.2) or omicron (B.1.1.529). The booster vaccine exhibited an acceptable safety profile. The overall frequencies of solicited local and systemic adverse events (evaluated in the safety subset, n=6067) were higher among vaccine recipients than placebo recipients after the primary and booster doses. The frequency of solicited adverse events in the Ad26.COV2.S group were similar following the primary and booster vaccinations (local adverse events, 1676 [55·6%] of 3015 vs 896 [57·5%] of 1559, respectively; systemic adverse events, 1764 [58·5%] of 3015 vs 821 [52·7%] of 1559, respectively). Solicited adverse events were transient and mostly grade 1-2 in severity. INTERPRETATION: A homologous Ad26.COV2.S booster administered 2 months after primary single-dose vaccination in adults had an acceptable safety profile and was efficacious against moderate to severe-critical COVID-19. Studies assessing efficacy against newer variants and with longer follow-up are needed. FUNDING: Janssen Research & Development.

Durable antibody responses elicited by 1 dose of Ad26.COV2.S and substantial increase after boosting: 2 randomized clinical trials.

BACKGROUND: Ad26.COV2.S is a well-tolerated and effective vaccine against COVID-19. We evaluated durability of anti-SARS-CoV-2 antibodies elicited by single-dose Ad26.COV2.S and the impact of boosting. METHODS: In randomized, double-blind, placebo-controlled, phase 1/2a and phase 2 trials, participants received single-dose Ad26.COV2.S (5 × 1010 viral particles [vp]) followed by booster doses of 5 × 1010 vp or 1.25 × 1010 vp. Neutralizing antibody levels were determined by a virus neutralization assay (VNA) approximately 8-9 months after dose 1. Binding and neutralizing antibody levels were evaluated by an enzyme-linked immunosorbent assay and pseudotyped VNA 6 months after dose 1 and 7 and 28 days after boosting. RESULTS: Data were analyzed from phase 1/2a participants enrolled from 22 July-18 December 2020 (Cohort 1a, 18-55 years [y], N = 25; Cohort 2a, 18-55y, N = 17; Cohort 3, ≥65y, N = 22), and phase 2 participants from 14 to 22 September 2020 (18-55y and ≥ 65y, N = 73). Single-dose Ad26.COV2.S elicited stable neutralizing antibodies for at least 8-9 months and stable binding antibodies for at least 6 months, irrespective of age. A 5 × 1010 vp 2-month booster dose increased binding antibodies by 4.9- to 6.2-fold 14 days post-boost versus 28 days after initial immunization. A 6-month booster elicited a steep and robust 9-fold increase in binding antibody levels 7 days post-boost. A 5.0-fold increase in neutralizing antibodies was observed by 28 days post-boost for the Beta variant. A 1.25 × 1010 vp 6-month booster elicited a 3.6-fold increase in binding antibody levels at 7 days post-boost versus pre-boost, with a similar magnitude of post-boost responses in both age groups. CONCLUSIONS: Single-dose Ad26.COV2.S elicited durable antibody responses for at least 8 months and elicited immune memory. Booster-elicited binding and neutralizing antibody responses were rapid and robust, even with a quarter vaccine dose, and stronger with a longer interval since primary vaccination. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04436276, NCT04535453.

Safety and immunogenicity of 3 formulations of a Sabin inactivated poliovirus vaccine produced on the PER.C6® cell line: A phase 2, double-blind, randomized, controlled study in infants vaccinated at 6, 10 and 14 weeks of age.

An inactivated poliovirus vaccine candidate using Sabin strains (sIPV) grown on the PER.C6® cell line was assessed in infants after demonstrated immunogenicity and safety in adults. The study recruited 300 infants who were randomized (1:1:1:1) to receive one of 3 dose levels of sIPV or a conventional IPV based on Salk strains (cIPV). Poliovirus-neutralizing antibodies were measured before the first dose and 28 days after the third dose. Reactogenicity was assessed for 7 days and unsolicited adverse events (AEs) for 28 days after each vaccination. Serious AEs (SAEs) were recorded throughout the study. Solicited AEs were mostly mild to moderate. None of the SAEs reported in the study were judged vaccine related, including one fatal SAE due to aspiration of vomitus that occurred 26 days after the third dose of low-dose sIPV. After 3 sIPV vaccinations and across all dose levels, seroconversion (SC) rates were at least 92% against Sabin poliovirus types and at least 80% against Salk types, with a dose-response in neutralizing antibody geometric mean titers (GMTs) observed across the 3 sIPV groups. Compared to cIPV, the 3 sIPV groups displayed similar or higher SC rates and GMTs against the 3 Sabin types but showed a lower response against Salk types 1 and 2; this was most visible for Salk type 1. While the PER.C6® cell line-based sIPV showed an acceptable safety profile and immunogenicity in infants, lower seroprotection against type 1 warrants optimization of dose level and additional clinical evaluation.

Final Analysis of Efficacy and Safety of Single-Dose Ad26.COV2.S.

BACKGROUND: The Ad26.COV2.S vaccine was highly effective against severe-critical coronavirus disease 2019 (Covid-19), hospitalization, and death in the primary phase 3 efficacy analysis. METHODS: We conducted the final analysis in the double-blind phase of our multinational, randomized, placebo-controlled trial, in which adults were assigned in a 1:1 ratio to receive single-dose Ad26.COV2.S (5×1010 viral particles) or placebo. The primary end points were vaccine efficacy against moderate to severe-critical Covid-19 with onset at least 14 days after administration and at least 28 days after administration in the per-protocol population. Safety and key secondary and exploratory end points were also assessed. RESULTS: Median follow-up in this analysis was 4 months; 8940 participants had at least 6 months of follow-up. In the per-protocol population (39,185 participants), vaccine efficacy against moderate to severe-critical Covid-19 at least 14 days after administration was 56.3% (95% confidence interval [CI], 51.3 to 60.8; 484 cases in the vaccine group vs. 1067 in the placebo group); at least 28 days after administration, vaccine efficacy was 52.9% (95% CI, 47.1 to 58.1; 433 cases in the vaccine group vs. 883 in the placebo group). Efficacy in the United States, primarily against the reference strain (B.1.D614G) and the B.1.1.7 (alpha) variant, was 69.7% (95% CI, 60.7 to 76.9); efficacy was reduced elsewhere against the P.1 (gamma), C.37 (lambda), and B.1.621 (mu) variants. Efficacy was 74.6% (95% CI, 64.7 to 82.1) against severe-critical Covid-19 (with only 4 severe-critical cases caused by the B.1.617.2 [delta] variant), 75.6% (95% CI, 54.3 to 88.0) against Covid-19 leading to medical intervention (including hospitalization), and 82.8% (95% CI, 40.5 to 96.8) against Covid-19-related death, with protection lasting 6 months or longer. Efficacy against any severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was 41.7% (95% CI, 36.3 to 46.7). Ad26.COV2.S was associated with mainly mild-to-moderate adverse events, and no new safety concerns were identified. CONCLUSIONS: A single dose of Ad26.COV2.S provided 52.9% protection against moderate to severe-critical Covid-19. Protection varied according to variant; higher protection was observed against severe Covid-19, medical intervention, and death than against other end points and lasted for 6 months or longer. (Funded by Janssen Research and Development and others; ENSEMBLE ClinicalTrials.gov number, NCT04505722.).

Safety and Efficacy of Single-Dose Ad26.COV2.S Vaccine against Covid-19.

BACKGROUND: The Ad26.COV2.S vaccine is a recombinant, replication-incompetent human adenovirus type 26 vector encoding full-length severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein in a prefusion-stabilized conformation. METHODS: In an international, randomized, double-blind, placebo-controlled, phase 3 trial, we randomly assigned adult participants in a 1:1 ratio to receive a single dose of Ad26.COV2.S (5×1010 viral particles) or placebo. The primary end points were vaccine efficacy against moderate to severe-critical coronavirus disease 2019 (Covid-19) with an onset at least 14 days and at least 28 days after administration among participants in the per-protocol population who had tested negative for SARS-CoV-2. Safety was also assessed. RESULTS: The per-protocol population included 19,630 SARS-CoV-2-negative participants who received Ad26.COV2.S and 19,691 who received placebo. Ad26.COV2.S protected against moderate to severe-critical Covid-19 with onset at least 14 days after administration (116 cases in the vaccine group vs. 348 in the placebo group; efficacy, 66.9%; adjusted 95% confidence interval [CI], 59.0 to 73.4) and at least 28 days after administration (66 vs. 193 cases; efficacy, 66.1%; adjusted 95% CI, 55.0 to 74.8). Vaccine efficacy was higher against severe-critical Covid-19 (76.7% [adjusted 95% CI, 54.6 to 89.1] for onset at ≥14 days and 85.4% [adjusted 95% CI, 54.2 to 96.9] for onset at ≥28 days). Despite 86 of 91 cases (94.5%) in South Africa with sequenced virus having the 20H/501Y.V2 variant, vaccine efficacy was 52.0% and 64.0% against moderate to severe-critical Covid-19 with onset at least 14 days and at least 28 days after administration, respectively, and efficacy against severe-critical Covid-19 was 73.1% and 81.7%, respectively. Reactogenicity was higher with Ad26.COV2.S than with placebo but was generally mild to moderate and transient. The incidence of serious adverse events was balanced between the two groups. Three deaths occurred in the vaccine group (none were Covid-19-related), and 16 in the placebo group (5 were Covid-19-related). CONCLUSIONS: A single dose of Ad26.COV2.S protected against symptomatic Covid-19 and asymptomatic SARS-CoV-2 infection and was effective against severe-critical disease, including hospitalization and death. Safety appeared to be similar to that in other phase 3 trials of Covid-19 vaccines. (Funded by Janssen Research and Development and others; ENSEMBLE ClinicalTrials.gov number, NCT04505722.).

Interim Results of a Phase 1-2a Trial of Ad26.COV2.S Covid-19 Vaccine.

BACKGROUND: Efficacious vaccines are urgently needed to contain the ongoing coronavirus disease 2019 (Covid-19) pandemic of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A candidate vaccine, Ad26.COV2.S, is a recombinant, replication-incompetent adenovirus serotype 26 (Ad26) vector encoding a full-length and stabilized SARS-CoV-2 spike protein. METHODS: In this multicenter, placebo-controlled, phase 1-2a trial, we randomly assigned healthy adults between the ages of 18 and 55 years (cohort 1) and those 65 years of age or older (cohort 3) to receive the Ad26.COV2.S vaccine at a dose of 5×1010 viral particles (low dose) or 1×1011 viral particles (high dose) per milliliter or placebo in a single-dose or two-dose schedule. Longer-term data comparing a single-dose regimen with a two-dose regimen are being collected in cohort 2; those results are not reported here. The primary end points were the safety and reactogenicity of each dose schedule. RESULTS: After the administration of the first vaccine dose in 805 participants in cohorts 1 and 3 and after the second dose in cohort 1, the most frequent solicited adverse events were fatigue, headache, myalgia, and injection-site pain. The most frequent systemic adverse event was fever. Systemic adverse events were less common in cohort 3 than in cohort 1 and in those who received the low vaccine dose than in those who received the high dose. Reactogenicity was lower after the second dose. Neutralizing-antibody titers against wild-type virus were detected in 90% or more of all participants on day 29 after the first vaccine dose (geometric mean titer [GMT], 212 to 354), regardless of vaccine dose or age group, and reached 96% by day 57 with a further increase in titers (GMT, 288 to 488) in cohort 1a. Titers remained stable until at least day 71. A second dose provided an increase in the titer by a factor of 2.6 to 2.9 (GMT, 827 to 1266). Spike-binding antibody responses were similar to neutralizing-antibody responses. On day 15, CD4+ T-cell responses were detected in 76 to 83% of the participants in cohort 1 and in 60 to 67% of those in cohort 3, with a clear skewing toward type 1 helper T cells. CD8+ T-cell responses were robust overall but lower in cohort 3. CONCLUSIONS: The safety and immunogenicity profiles of Ad26.COV2.S support further development of this vaccine candidate. (Funded by Johnson & Johnson and the Biomedical Advanced Research and Development Authority of the Department of Health and Human Services; COV1001 ClinicalTrials.gov number, NCT04436276.).

Oral HPV16 DNA as a screening tool to detect early oropharyngeal squamous cell carcinoma.

Given that oropharyngeal squamous cell carcinoma (OPSCC) have now surpassed cervical cancer as the most common human papillomavirus (HPV)-driven cancer, there is an interest in developing non-invasive predictive biomarkers to early detect HPV-driven OPSCC. In total, 665 cancer-free individuals were recruited from Queensland, Australia. Oral HPV16 DNA positivity in those individuals was determined by our in-house developed sensitive PCR method. Individuals with (n = 9) or without (n = 12) oral HPV16 infections at baseline were followed for a median duration of 24 mo. Individuals with persistent oral HPV16 infection (≥ 30 mo) were invited for clinical examination of their oral cavity and oropharynx by an otolaryngologist. Oral HPV16 DNA was detected in 12 out of 650 cancer-free individuals (1.8%; 95% confidence interval [CI]: 1.0-3.2). Of the 3 individuals with persistent oral HPV16 infection, the first individual showed no clinical evidence of pathology. The second individual was diagnosed with a 2 mm invasive squamous cell carcinoma (T1N0M0) positive for both p16INK4a expression and HPV16 DNA. The third individual was found to have a mildly dysplastic lesion in the tonsillar region that was negative for p16INK4a expression and HPV16 DNA and she continues to have HPV16 DNA in her saliva. Taken together, our data support the value of using an oral HPV16 DNA assay as a potential screening tool for the detection of microscopic HPV-driven OPSCC. Larger multicenter studies across various geographic regions recruiting populations at a higher risk of developing HPV-driven OPSCC are warranted to extend and confirm the results of the current investigation.

Oral HPV16 Prevalence in Oral Potentially Malignant Disorders and Oral Cavity Cancers.

The role of human papillomavirus type 16 (HPV16) in oral potentially malignant disorders (OPMD) and oral cavity carcinoma (OC) is still under debate. We investigated HPV16 prevalence in unstimulated saliva, oral rinse samples, oral swabs and tumour biopsies collected from OPMD (n = 83) and OC (n = 106) patients. HPV16 genotype, viral load, physical status (episomal vs. integrated) and tumour p16INK4a expression were determined. Oral HPV16 prevalence was higher in OC than in OPMD, but this difference was not statistically significant (7.5% (8/106) versus 3.6% (3/83), odds ratio (OR): 2.18, 95% confidence interval (CI): 0.56, 8.48, p = 0.26). There was a significant association (p < 0.05) between oral HPV16 infection and heavy tobacco consumption. Real-time PCR results indicated that no integration events occurred in either OPMD or OC cases based on the HPV16 E2/E6 ratio. HPV16 positive OPMD and OC patients had similar HPV16 E2 and E6 viral loads. The inter-rater agreement between tumour p16INK4a expression and oral HPV16 infection was considered as fair (k = 0.361) for OC. Our data suggest that the involvement of HPV16 in oral carcinogenesis is limited.

Unlocking the Potential of Saliva-Based Test to Detect HPV-16-Driven Oropharyngeal Cancer.

The incidence of human papillomavirus (HPV)-positive oropharyngeal cancer (OPC) is rising in high-income countries, including Australia. Increasing evidence suggests that accurate HPV testing is pivotal for clinical decision making and treatment planning in these patients. Recently, the eighth edition of the American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC) tumor-node-metastasis (TNM) staging system for OPC (based on the p16INK4a (p16) status) was proposed and has been implemented. However, the applicability of this new staging system is still far from clear. In our study, n = 127 OPC patients from Queensland, Australia were recruited, and the tumor p16 expression in these patients was examined using immunohistochemical (IHC) analysis. HPV-16 genotyping, viral load, and physical status (episomal versus integrated) in the saliva samples of OPC patients were determined using the qPCR method. A good inter-rater agreement (k = 0.612) was found between tumor p16 expression and oral HPV-16 infection in OPC. Importantly, according to the eighth edition staging system, HPV-16 DNA viral load (>10 copies/50 ng) was significantly associated with the advanced stages of OPC. In concordance with previous studies, a mixed HPV-16 form (partially or fully integrated) was predominately found in OPC patients. Taken together, our data support HPV-16 detection in saliva as a screening biomarker to identify people within the community who are at risk of developing OPC.

Adenovirus based HPV L2 vaccine induces broad cross-reactive humoral immune responses.

Oncogenic high-risk human papillomavirus (HPV) infections cause a substantial number of genital and non-genital cancers worldwide. Approximately 70% of all cervical cancers are caused by the high-risk HPV16 and 18 types. The remaining 30% can be attributed to twelve other high-risk HPV-types. Highly efficacious 2-valent, 4-valent and 9-valent L1 protein based prophylactic HPV vaccines are available however with limited cross-protection. To further increase the coverage, development of a multivalent cross-protective HPV vaccine is currently focused on the conserved N-terminus of HPV's L2 protein. We have developed a vaccine candidate based on the rare human adenovirus type 35 (HAdV35) vector that displays a concatemer of L2 protein epitopes from four different HPV-types via protein IX (pIX). A mix of two heterologous HAdV35 pIX-L2 display vectors present highly conserved linear epitopes of nine HPV-types. Each HAdV35 pIX-L2 display vector exhibits a good manufacturability profile. HAdV35 pIX-L2 display vaccine vectors were immunogenic and induced neutralizing antibodies against HPV-types included in the vaccine and cross-neutralizing antibodies against distant a HPV-type not included in the vaccine in mice. The HAdV35 pIX-L2 display vectors offer an opportunity for a multivalent HAdV-based prophylactic HPV vaccine.

Adenovirus vector-based prime-boost vaccination via heterologous routes induces cervicovaginal CD8+ T cell responses against HPV16 oncoproteins.

Recent advances in immunotherapy against cancer underscore the importance of T lymphocytes and tumor microenvironment, but few vaccines targeting cancer have been approved likely due in part to the dearth of common tumor antigens, insufficient immunogenicity and the evolution of immune evasion mechanisms during the progression to malignancy. Human papillomaviruses (HPVs) are the primary etiologic agents of cervical cancer and progression from persistent HPV-infection to cervical intraepithelial lesions and eventually cancer requires persistent expression of the oncoproteins E6 and E7. This offers the opportunity to specifically target these virus-specific antigens for vaccine-induced clearance of infected cells before cancers develop. Here we have evaluated the immunogenicity of Adenovirus Types 26 and 35 derived vectors expressing a fusion of HPV16 E6 and E7 oncoproteins after intramuscular (IM) and/or intravaginal (Ivag) immunization in mice. The adenovirus vectors were shown to transduce an intact cervicovaginal epithelium. IM prime followed by Ivag boost maximized the induction and trafficking of HPV-specific CD8+ T cells producing IFN-γ and TNF-α to the cervicovaginal tract. Importantly, the cervicovaginal CD8+ T cells expressed CD69 and CD103; hallmarks of intraepithelial tissue-resident memory CD8+ T cells. This prime-boost strategy targeting heterologous locations also induced circulating HPV-specific CD8+ T cell responses. Our study prompts further evaluation of Ivag immunization with adenoviral vectors expressing modified E6 and E7 antigens for therapeutic vaccination against persistent HPV infection and cervical intraepithelial neoplasia.

Development of a replication-deficient adenoviral vector-based vaccine candidate for the interception of HPV16- and HPV18-induced infections and disease.

High-risk Human papilloma virus (HPV) types are the causative agents of cervical cancer and several other anogenital malignancies. The viral proteins expressed in the (pre)malignant cells are considered ideal targets for immunological intervention. Many approaches have been evaluated for this purpose, mostly aiming at the induction of HPV16 E7- and/or E6-specific cellular immunogenicity. As clinical success has so far been limited, novel approaches are required. We describe the development and pre-clinical testing of a vaccine candidate consisting of replication-deficient adenovirus type 26 and 35 based vectors for the interception of HPV16- and HPV18-related disease. We developed HPV16- and HPV18-specific antigens consisting of fusion proteins of E2, E6 and E7. The vaccine will be suitable for every disease stage, from incident and persistent infections where E2 is predominantly expressed up to late stages where E6 and E7 expression are upregulated. Importantly E6 and E7 are present as reordered fragments to abrogate the transforming activity of these two proteins. Loss of transforming activity was demonstrated in different in vitro models. Robust T-cell immunogenicity was induced upon immunization of mice with the vaccine candidate. Finally, the developed vaccine vectors showed considerable therapeutic efficacy in the TC-1 mouse model. The absence of transforming activity of the antigens and the favorable immunogenicity profile of the adenovirus based vectors along with the fact that these vectors can be readily produced on a large scale makes this approach attractive for clinical evaluation.

Astrocytes are central in the pathomechanisms of vanishing white matter.

Vanishing white matter (VWM) is a fatal leukodystrophy that is caused by mutations in genes encoding subunits of eukaryotic translation initiation factor 2B (eIF2B). Disease onset and severity are codetermined by genotype. White matter astrocytes and oligodendrocytes are almost exclusively affected; however, the mechanisms of VWM development remain unclear. Here, we used VWM mouse models, patients' tissue, and cell cultures to investigate whether astrocytes or oligodendrocytes are the primary affected cell type. We generated 2 mouse models with mutations (Eif2b5Arg191His/Arg191His and Eif2b4Arg484Trp/Arg484Trp) that cause severe VWM in humans and then crossed these strains to develop mice with various mutation combinations. Phenotypic severity was highly variable and dependent on genotype, reproducing the clinical spectrum of human VWM. In all mutant strains, impaired maturation of white matter astrocytes preceded onset and paralleled disease severity and progression. Bergmann glia and retinal Müller cells, nonforebrain astrocytes that have not been associated with VWM, were also affected, and involvement of these cells was confirmed in VWM patients. In coculture, VWM astrocytes secreted factors that inhibited oligodendrocyte maturation, whereas WT astrocytes allowed normal maturation of VWM oligodendrocytes. These studies demonstrate that astrocytes are central in VWM pathomechanisms and constitute potential therapeutic targets. Importantly, astrocytes should also be considered in the pathophysiology of other white matter disorders.

Mice with megalencephalic leukoencephalopathy with cysts: a developmental angle.

OBJECTIVE: Megalencephalic leukoencephalopathy with cysts (MLC) is a genetic disease characterized by infantile onset white matter edema and delayed onset neurological deterioration. Loss of MLC1 function causes MLC. MLC1 is involved in ion-water homeostasis, but its exact role is unknown. We generated Mlc1-null mice for further studies. METHODS: We investigated which brain cell types express MLC1, compared developmental expression in mice and men, and studied the consequences of loss of MLC1 in Mlc1-null mice. RESULTS: Like humans, mice expressed MLC1 only in astrocytes, especially those facing fluid-brain barriers. In mice, MLC1 expression increased until 3 weeks and then stabilized. In humans, MLC1 expression was highest in the first year, decreased, and stabilized from approximately 5 years. Mlc1-null mice had early onset megalencephaly and increased brain water content. From 3 weeks, abnormal astrocytes were present with swollen processes abutting fluid-brain barriers. From 3 months, widespread white matter vacuolization with intramyelinic edema developed. Mlc1-null astrocytes showed slowed regulatory volume decrease and reduced volume-regulated anion currents, which increased upon MLC1 re-expression. Mlc1-null astrocytes showed reduced expression of adhesion molecule GlialCAM and chloride channel ClC-2, but no substantial changes in other known MLC1-interacting proteins. INTERPRETATION: Mlc1-null mice replicate early stages of the human disease with early onset intramyelinic edema. The cellular functional defects, described for human MLC, were confirmed. The earliest change was astrocytic swelling, substantiating that in MLC the primary defect is in volume regulation by astrocytes. MLC1 expression affects expression of GlialCAM and ClC-2. Abnormal interplay between these proteins is part of the pathomechanisms of MLC.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation: clinical and genetic characterization and target for therapy.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation is a disorder caused by recessive mutations in the gene DARS2, which encodes mitochondrial aspartyl-tRNA synthetase. Recent observations indicate that the phenotypic range of the disease is much wider than initially thought. Currently, no treatment is available. The aims of our study were (i) to explore a possible genotype-phenotype correlation; and (ii) to identify potential therapeutic agents that modulate the splice site mutations in intron 2 of DARS2, present in almost all patients. A cross-sectional observational study was performed in 78 patients with two DARS2 mutations in the Amsterdam and Helsinki databases up to December 2012. Clinical information was collected via questionnaires. An inventory was made of the DARS2 mutations in these patients and those previously published. An assay was developed to assess mitochondrial aspartyl-tRNA synthetase enzyme activity in cells. Using a fluorescence reporter system we screened for drugs that modulate DARS2 splicing. Clinical information of 66 patients was obtained. The clinical severity varied from infantile onset, rapidly fatal disease to adult onset, slow and mild disease. The most common phenotype was characterized by childhood onset and slow neurological deterioration. Full wheelchair dependency was rare and usually began in adulthood. In total, 60 different DARS2 mutations were identified, 13 of which have not been reported before. Except for 4 of 42 cases published by others, all patients were compound heterozygous. Ninety-four per cent of the patients had a splice site mutation in intron 2. The groups of patients sharing the same two mutations were too small for formal assessment of genotype-phenotype correlation. However, some combinations of mutations were consistently associated with a mild phenotype. The mitochondrial aspartyl-tRNA synthetase activity was strongly reduced in patient cells. Among the compounds screened, cantharidin was identified as the most potent modulator of DARS2 splicing. In conclusion, the phenotypic spectrum of leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation is wide, but most often the disease has a relatively slow and mild course. The available evidence suggests that the genotype influences the phenotype, but because of the high number of private mutations, larger numbers of patients are necessary to confirm this. The activity of mitochondrial aspartyl-tRNA synthetase is significantly reduced in patient cells. A compound screen established a 'proof of principle' that the splice site mutation can be influenced. This finding is promising for future therapeutic strategies.

Released selective pressure on a structural domain gives new insights on the functional relaxation of mitochondrial aspartyl-tRNA synthetase.

Mammalian mitochondrial aminoacyl-tRNA synthetases are nuclear-encoded enzymes that are essential for mitochondrial protein synthesis. Due to an endosymbiotic origin of the mitochondria, many of them share structural domains with homologous bacterial enzymes of same specificity. This is also the case for human mitochondrial aspartyl-tRNA synthetase (AspRS) that shares the so-called bacterial insertion domain with bacterial homologs. The function of this domain in the mitochondrial proteins is unclear. Here, we show by bioinformatic analyses that the sequences coding for the bacterial insertion domain are less conserved in opisthokont and protist than in bacteria and viridiplantae. The divergence suggests a loss of evolutionary pressure on this domain for non-plant mitochondrial AspRSs. This discovery is further connected with the herein described occurrence of alternatively spliced transcripts of the mRNAs coding for some mammalian mitochondrial AspRSs. Interestingly, the spliced transcripts alternately lack one of the four exons that code for the bacterial insertion domain. Although we showed that the human alternative transcript is present in all tested tissues; co-exists with the full-length form, possesses 5'- and 3'-UTRs, a poly-A tail and is bound to polysomes, we were unable to detect the corresponding protein. The relaxed selective pressure combined with the occurrence of alternative splicing, involving a single structural sub-domain, favors the hypothesis of the loss of function of this domain for AspRSs of mitochondrial location. This evolutionary divergence is in line with other characteristics, established for the human mt-AspRS, that indicate a functional relaxation of non-viridiplantae mt-AspRSs when compared to bacterial and plant ones, despite their common ancestry.

A yeast purification system for human translation initiation factors eIF2 and eIF2Bε and their use in the diagnosis of CACH/VWM disease.

Recessive inherited mutations in any of five subunits of the general protein synthesis factor eIF2B are responsible for a white mater neurodegenerative disease with a large clinical spectrum. The classical form is called Childhood Ataxia with CNS hypomyelination (CACH) or Vanishing White Matter Leukoencephalopathy (VWM). eIF2B-related disorders affect glial cells, despite the fact that eIF2B is a ubiquitous protein that functions as a guanine-nucleotide exchange factor (GEF) for its partner protein eIF2 in the translation initiation process in all eukaryotic cells. Decreased eIF2B activity measured by a GEF assay in patients' immortalised lymphocytic cells provides a biochemical diagnostic assay but is limited by the availability of eIF2 protein, which is classically purified from a mammalian cell source by column chromatography. Here we describe the generation of a recombinant expression system to produce purified human eIF2 from yeast cells. We demonstrate that human eIF2 can function in yeast cells in place of the equivalent yeast factor. We purify human eIF2 and the C-terminal domain of human eIF2Bε using affinity chromatography from engineered yeast cells and find that both function in a GEF assay: the first demonstration that this human eIF2Bε domain has GEF function. We show that CACH/VWM mutations within this domain reduce its activity. Finally we demonstrate that the recombinant eIF2 functions similarly to eIF2 purified from rat liver in GEF assays with CACH/VWM eIF2B-mutated patient derived lymphocytic cells.

Pathogenic mutations causing LBSL affect mitochondrial aspartyl-tRNA synthetase in diverse ways.

The autosomal recessive white matter disorder LBSL (leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation) is caused by mutations in DARS2, coding for mtAspRS (mitochondrial aspartyl-tRNA synthetase). Generally, patients are compound heterozygous for mutations in DARS2. Many different mutations have been identified in patients, including several missense mutations. In the present study, we have examined the effects of missense mutations found in LBSL patients on the expression, enzyme activity, localization and dimerization of mtAspRS, which is important for understanding the cellular defect underlying the pathogenesis of the disease. Nine different missense mutations were analysed and were shown to have various effects on mtAspRS properties. Several mutations have a direct effect on the catalytic activity of the enzyme; others have an effect on protein expression or dimerization. Most mutations have a clear impact on at least one of the properties of mtAspRS studied, probably resulting in a small contribution of the missense variants to the mitochondrial aspartylation activity in the cell.

Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation is associated with cell-type-dependent splicing of mtAspRS mRNA.

LBSL (leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation) is an autosomal recessive white matter disorder with slowly progressive cerebellar ataxia, spasticity and dorsal column dysfunction. Magnetic resonance imaging shows characteristic abnormalities in the cerebral white matter and specific brain stem and spinal cord tracts. LBSL is caused by mutations in the gene DARS2, which encodes mtAspRS (mitochondrial aspartyl-tRNA synthetase). The selective involvement of specific white matter tracts in LBSL is striking since this protein is ubiquitously expressed. Almost all LBSL patients have one mutation in intron 2 of DARS2, affecting the splicing of the third exon. Using a splicing reporter construct, we find cell-type-specific differences in the sensitivity to these mutations: the mutations have a larger effect on exon 3 exclusion in neural cell lines, especially neuronal cell lines, than in non-neural cell lines. Furthermore, correct inclusion of exon 3 in the normal mtAspRS mRNA occurs less efficiently in neural cells than in other cell types, and this effect is again most pronounced in neuronal cells. The combined result of these two effects may explain the selective vulnerability of specific white matter tracts in LBSL patients.