Association of a Third Dose of BNT162b2 Vaccine With Incidence of SARS-CoV-2 Infection Among Health Care Workers in Israel.

Importance: Administration of a BNT162b2 booster dose (Pfizer-BioNTech) to fully vaccinated individuals aged 60 years and older was significantly associated with lower risk of SARS-CoV-2 infection and severe illness. Data are lacking on the effectiveness of booster doses for younger individuals and health care workers. Objective: To estimate the association of a BNT162b2 booster dose with SARS-CoV-2 infections among health care workers who were previously vaccinated with a 2-dose series of BNT162b2. Design, Setting, and Participants: This was a prospective cohort study conducted at a tertiary medical center in Tel Aviv, Israel. The study cohort included 1928 immunocompetent health care workers who were previously vaccinated with a 2-dose series of BNT162b2, and had enrolled between August 8 and 19, 2021, with final follow-up reported through September 20, 2021. Screening for SARS-CoV-2 infection was performed every 14 days. Anti-spike protein receptor binding domain IgG titers were determined at baseline and 1 month after enrollment. Cox regression with time-dependent analysis was used to estimate hazard ratios of SARS-CoV-2 infection between booster-immunized status and 2-dose vaccinated (booster-nonimmunized) status. Exposures: Vaccination with a booster dose of BNT162b2 vaccine. Main Outcomes and Measures: The primary outcome was SARS-CoV-2 infection, as confirmed by reverse transcriptase-polymerase chain reaction. Results: Among 1928 participants, the median age was 44 years (IQR, 36-52 years) and 1381 were women (71.6%). Participants completed the 2-dose vaccination series a median of 210 days (IQR, 205-213 days) before study enrollment. A total of 1650 participants (85.6%) received the booster dose. During a median follow-up of 39 days (IQR, 35-41 days), SARS-CoV-2 infection occurred in 44 participants (incidence rate, 60.2 per 100 000 person-days); 31 (70.5%) were symptomatic. Five SARS-CoV-2 infections occurred in booster-immunized participants and 39 in booster-nonimmunized participants (incidence rate, 12.8 vs 116 per 100 000 person-days, respectively). In a time-dependent Cox regression analysis, the adjusted hazard ratio of SARS-CoV-2 infection for booster-immunized vs booster-nonimmunized participants was 0.07 (95% CI, 0.02-0.20). Conclusions and Relevance: Among health care workers at a single center in Israel who were previously vaccinated with a 2-dose series of BNT162b2, administration of a booster dose compared with not receiving one was associated with a significantly lower rate of SARS-CoV-2 infection over a median of 39 days of follow-up. Ongoing surveillance is required to assess durability of the findings.

CD59 deficiency is associated with chronic hemolysis and childhood relapsing immune-mediated polyneuropathy.

CD59 deficiency is a common finding in RBCs and WBCs in patients with chronic hemolysis suffering from paroxysmal nocturnal hemoglobinuria in which the acquired mutation in the PIGA gene leads to membrane loss of glycosylphosphatidylinositol-anchored membrane proteins, including CD59. The objective of the present study was to elucidate the molecular basis of childhood familial chronic Coombs-negative hemolysis and relapsing polyneuropathy presenting as chronic inflammatory demyelinating polyradiculoneuropathy in infants of North-African Jewish origin from 4 unrelated families. A founder mutation was searched for using homozygosity mapping followed by exome sequencing. The expression of CD59, CD55, and CD14 was examined in blood cells by flow cytometry followed by Western blot of the CD59 protein. A homozygous missense mutation, p.Cys89Tyr in CD59, was identified in all patients. The mutation segregated with the disease in the families and had a carrier rate of 1:66 among Jewish subjects of North-African origin. The mutated protein was present in the patients' cells in reduced amounts and was undetectable on the membrane surface. Based on the results of the present study, we conclude that the Cys89Tyr mutation in CD59 is associated with a failure of proper localization of the CD59 protein in the cell surface. This mutation is manifested clinically in infancy by chronic hemolysis and relapsing peripheral demyelinating disease.

Relationship among sleep, work features, and SARS-cov-2 vaccine antibody response in hospital workers.

STUDY OBJECTIVES: Immunity is influenced by sleep and the circadian rhythm. Healthcare workers are predisposed to both insufficient sleep and circadian disruption. This study aimed to evaluate the relationship between sleep and work characteristics and the antibody response to the mRNA SARS-CoV-2 vaccine BNT162b2. METHODS: The authors' prospective cohort study ("COVI3") evaluated the effect of a third (booster) dose of the BNT162b2 vaccine. A subset of participants provided information on anthropometric measures, sleep, stress and work characteristics including shift work and number of work hours per week. Blood samples for anti-S1-RBD IgG antibody levels were obtained 21 weeks following receipt of the third dose of the vaccine. RESULTS: In total, 201 healthcare workers (73% women) were included. After adjustment for age, body mass index (BMI), shift work, smoking status, and perceived stress, short sleep duration (<7 h per night) was associated with lower anti-S1-RBD IgG levels (Odds ratio 2.36 [95% confidence interval 1.08-5.13]). Participants who performed shift work had higher odds of lower anti-S1-RBD IgG levels compared to those who did not work in shifts [odds ratio = 2.99 (95% confidence interval 1.40, 6.39)] after accounting for age, short sleep duration, BMI, smoking status and perceived stress. CONCLUSIONS: Shift work and self-reported short sleep duration were associated with a lower antibody response following a booster dose of the SARS-CoV-2 vaccine. These findings suggest that the efficacy of vaccination, particularly among healthcare workers, may be augmented by addressing both sleep and circadian alignment.

An outbreak of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infections among hospital personnel with high mRNA vaccine uptake.

Real-world studies have demonstrated impressive effectiveness of the BNT162b2 COVID-19 vaccine in preventing symptomatic and asymptomatic SARS-CoV-2 infection. We describe an outbreak of SARS-CoV-2 infections in a hospital with high vaccine uptake. We found a low secondary attack rate (7%), suggesting low infectivity of vaccinated persons with vaccine breakthrough SARS-CoV-2 infections.

Persistent ER stress induces the spliced leader RNA silencing pathway (SLS), leading to programmed cell death in Trypanosoma brucei.

Trypanosomes are parasites that cycle between the insect host (procyclic form) and mammalian host (bloodstream form). These parasites lack conventional transcription regulation, including factors that induce the unfolded protein response (UPR). However, they possess a stress response mechanism, the spliced leader RNA silencing (SLS) pathway. SLS elicits shut-off of spliced leader RNA (SL RNA) transcription by perturbing the binding of the transcription factor tSNAP42 to its cognate promoter, thus eliminating trans-splicing of all mRNAs. Induction of endoplasmic reticulum (ER) stress in procyclic trypanosomes elicits changes in the transcriptome similar to those induced by conventional UPR found in other eukaryotes. The mechanism of up-regulation under ER stress is dependent on differential stabilization of mRNAs. The transcriptome changes are accompanied by ER dilation and elevation in the ER chaperone, BiP. Prolonged ER stress induces SLS pathway. RNAi silencing of SEC63, a factor that participates in protein translocation across the ER membrane, or SEC61, the translocation channel, also induces SLS. Silencing of these genes or prolonged ER stress led to programmed cell death (PCD), evident by exposure of phosphatidyl serine, DNA laddering, increase in reactive oxygen species (ROS) production, increase in cytoplasmic Ca(2+), and decrease in mitochondrial membrane potential, as well as typical morphological changes observed by transmission electron microscopy (TEM). ER stress response is also induced in the bloodstream form and if the stress persists it leads to SLS. We propose that prolonged ER stress induces SLS, which serves as a unique death pathway, replacing the conventional caspase-mediated PCD observed in higher eukaryotes.

The association of maternal SARS-CoV-2 vaccination-to-delivery interval and the levels of maternal and cord blood antibodies.

OBJECTIVE: To evaluate the correlation of maternal and cord blood levels of SARS-CoV-2 antibodies in pregnant women immunized against COVID-19. METHODS: A prospective cohort study was performed of pregnant women who delivered at a single university affiliated tertiary medical center. Women who received the COVID-19 vaccine (BNT162b2 Pfizer©) were approached. The correlation between levels of maternal sera and umbilical cord SARS-CoV-2 specific IgG was assessed. RESULTS: Overall, 58 women were included; of them, 19 had received a single dose and 39 received two doses of the COVID-19 vaccine. Positive levels of umbilical cord IgG were found in 13/19 (68.4%) and 38/39 (97.4%) women after the administration of a single dose and two doses of the vaccine, respectively. The levels of SARS-CoV-2 IgG antibodies in the maternal sera of vaccinated women were positively correlated to their respective concentrations in cord blood sera (ρ = 0.857; R2 linear = 0.719; P < 0.001). Thirteen days after vaccination, the ratio of maternal-to-umbilical cord anti Spike IgG antibodies was approximately 1, indicating relatively similar levels in maternal and cord sera. CONCLUSION: After the SARS-CoV-2 vaccine, levels of maternal and cord blood antibodies were positively correlated, especially when tested after 13 days following administration of the first dose of the vaccine.

The effect of a third-dose BNT162b2 vaccine on anti-SARS-CoV-2 antibody levels in immunosuppressed patients.

OBJECTIVES: The recent surge in coronavirus disease 2019 cases led to the consideration of a booster vaccine in previously vaccinated immunosuppressed individuals. However, the immunogenic effect of a third-dose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in immunosuppressed patients is still unknown. METHODS: This was an observational cohort study of 279 previously vaccinated immunosuppressed patients followed at a single tertiary hospital in Israel. Patients were administered a third dose of the Pfizer-BioNTech mRNA vaccine (BNT162b2) between July 14 and July 21, 2021. Levels of IgG antibodies against the spike receptor-binding domain of SARS-CoV-2 were measured 3 to 4 weeks after vaccination. RESULTS: Of the cohort of 279 patients, 124 (44.4%) had haematologic malignancies, 57 (20.4%) had rheumatologic diseases, and 98 (35.1%) were solid organ-transplant recipients. Anti-SARS-CoV-2 antibody levels increased in 74.9% of cases. Across the entire cohort, the median absolute antibody levels (expressed in AU/mL) increased from 7 (interquartile range (IQR), 0.1-69) to 243 (IQR, 2-4749) after the booster dose. The response significantly varied across subgroups: The transplant cohort showed the greatest increase in absolute antibody levels (from 52 (IQR, 7.25-184.5) to 1824 (IQR, 161-9686)), followed by the rheumatology (from 22 (IQR, 1-106) to 1291 (IQR, 6-6231)) and haemato-oncology (from 1 (IQR, 0.1-7) to 7.5 (IQR, 0.1-407.5)) cohorts. The χ2 test was 8.30 for difference in fold change (p = 0.016). Of the 193 patients who were seronegative at baseline, 76 became seropositive after vaccination, corresponding to a 39.4% (95% CI, 32.8%-46.4%) seroconversion rate. Transplant patients had the highest seroconversion rate (58.3% (95% CI, 44.3%-71.2%)), followed by rheumatology (44.1% (95% CI, 28.9%-60.5%)) and haemato-oncology (29.7% (95% CI, 22%-38.8%); χ2 = 11.87; p = 0.003) patients. DISCUSSION: A third dose of BNT162b2 is immunogenic in most immunosuppressed individuals, although antibody response may differ based on the type of disease and immunosuppression. The antibody level that correlates with protection is still unknown; thus, future studies are needed to evaluate clinical outcomes.

Trypanosome spliced-leader-associated RNA (SLA1) localization and implications for spliced-leader RNA biogenesis.

Spliced-leader-associated RNA (SLA1) guides the pseudouridylation at position -12 (relative to the 5' splice site) of the spliced-leader (SL) RNA in all trypanosomatid species. Nevertheless, the exact role of this RNA is currently unknown. Here, we demonstrate that the absence of pseudouridine on Leptomonas collosoma SL RNA has only a minor effect on the ability of this RNA to function in trans splicing in vivo. To investigate the possible role of SLA1 during SL RNA biogenesis, the structure of the SL RNA was examined in permeable Trypanosoma brucei cells depleted for CBF5, the H/ACA pseudouridine synthase, lacking SLA1. Our results suggest that in the absence of SLA1, the SL RNA secondary structure is changed, as was detected by differential sensitivity to oligonucleotide-directed RNase H cleavage, suggesting that the association of SLA1 maintains the SL RNA in a structural form which is distinct from the structure of the SL RNA in the steady state. In T. brucei cells depleted for the SL RNA core protein SmD1, SL RNA first accumulates in large amounts in the nucleus and then is expelled to the cytoplasm. Here, we demonstrate by in vivo aminomethyltrimethyl UV cross-linking studies that under SmD1 depletion, SLA1 remains bound to SL RNA and escorts the SL RNA to the cytoplasm. In situ hybridization with SLA1 and SL RNA demonstrates colocalization between SLA1 and the SL RNA transcription factor tSNAP42, as well as with Sm proteins, suggesting that SLA1 associates with SL RNA early in its biogenesis. These results demonstrate that SLA1 is a unique chaperonic RNA that functions during the early biogenesis of SL RNA to maintain a structure that is most probably suitable for cap 4 modification.

Treatment inferred from mutations identified using massive parallel sequencing leads to clinical benefit in some heavily pretreated cancer patients.

Molecular portraits of numerous tumors have flooded oncologists with vast amounts of data. In parallel, effective inhibitors of central pathways have shown great clinical benefit. Together, this promises potential clinical benefits to otherwise end-stage cancer patients. Here, we report a clinical service offering mutation detection of archived samples using the ion Ampliseq cancer panel coupled with clinical consultation.A multidisciplinary think tank consisting of oncologists, molecular-biologists, genetic counselors, and pathologists discussed 67 heavily pretreated, advanced cancer patient cases, taking into account mutations identified using ion Ampliseq cancer panel, medical history, and relevant literature.The team generated a treatment plan, targeting specific mutations, for 41 out of 64 cases. Three patients died before results were available. For 32 patients, the treating oncologists chose not to include the panel recommendation in the treatment plan for various reasons. Nine patients were treated as recommended by the panel, 5 with clinical benefit, and 4 with disease progression.This study suggests that routine use of massive parallel tumor sequencing is feasible and can judiciously affect treatment decisions when coupled with multidisciplinary team-based decision making. Administration of personalized based therapies at an earlier stage of disease, expansion of genetic alterations examined, and increased availability of targeted therapies may lead to further improvement in the clinical outcome of metastatic cancer patients.

Devastating recurrent brain ischemic infarctions and retinal disease in pediatric patients with CD59 deficiency.

Identification of CD59 p.Cys89Tyr mutation in 5 patients from North-African Jewish origin presenting with chronic inflammatory demyelinating polyradiculoneuropathy like disease and chronic hemolysis, led us to reinvestigate an unsolved disease in 2 siblings from the same origin who died 17 years ago. The two patients carried the same CD59 gene mutation previously described by our group. These children had quiet similar disease course but in addition developed devastating recurrent brain infarctions, retinal and optic nerve involvement. Revising the brain autopsy of one of these patients confirmed the finding of multiple brain infarctions of different ages. CD59 protein expression was missing on brain endothelial cells by immunohistochemical staining. This new data expands the clinical spectrum of CD59 mutations and further emphasizes the need for its early detection and treatment.

Lynch Syndrome in high risk Ashkenazi Jews in Israel.

Lynch Syndrome is caused by mutations in DNA mismatch repair genes. Diagnosis is not always trivial and may be costly. Information regarding incidence, genotype-phenotype correlation, spectrum of mutations and genes involved in specific populations facilitate the diagnostic process and contribute to clinical work-up. To report gene distribution, mutations detected and co-occurrence of related syndromes in a cohort of Ashkenazi Jews in Israel. Patients were identified in dedicated high risk clinics in 3 medical centers in Israel. Diagnostic process followed a multi-step scheme. It included testing for founder mutations, tumor testing, gene sequencing and MLPA. Lynch Syndrome was defined either by positive mutation testing, or by clinical criteria and positive tumor analysis. We report a cohort of 75 Ashkenazi families suspected of Lynch Syndrome. Mutations were identified in 51/75 (68%) families: 38 in MSH2, 9 in MSH6, and 4 in MLH1. 37/51 (73%) of these families carried one of the 3 'Ashkenazi' founder mutations in MSH2 or MSH6. Each of the other 14 families carried a private mutation. 3 (6%) were large deletions. Only 20/51 (39%) families were Amsterdam Criteria positive; 42 (82%) were positive for the Bethesda guidelines and 9 (18%) did not fulfill any Lynch Syndrome criteria. We report C-MMRD and co-occurrence of BRCA and Lynch Syndrome in our cohort. Mutation spectra and gene distribution among Ashkenazi Jews are unique. Three founder Lynch Syndrome mutations are found in 73% families with known mutations. Among the three, MSH2 and MSH6 are the most common. These features affect the phenotype, the diagnostic process, risk estimation, and genetic counseling.

Induction of ER stress response leading to programmed cell death in Trypanosoma brucei.

Trypanosomes are parasitic protozoans that include several medically and a variety of economically important parasites, such as Trypanosoma brucei, the causative agent of sleeping sickness. This parasite cycles between the insect host (procyclic form) and mammalian host (bloodstream form). These parasites lack transcription regulation, including factors that govern the unfolded protein response (UPR) in other eukaryotes. Gene expression is controlled posttranscriptionally by unique mechanisms such as trans-splicing and RNA editing and by mRNA stability. In trans-splicing, a common exon, the spliced leader (SL) is donated to all mRNAs from a small RNA, the SL RNA. The SL RNA is transcribed from a defined promoter assisted by the tSNAP complex. Despite the lack of transcriptional regulation, induction of ER stress elicits changes in the transcriptome similar to those induced by conventional UPR found in other eukaryotes. The mechanism of upregulation under UPR is dependent on differential stabilization of mRNAs. The transcriptome changes result in ER expansion and elevation in the ER chaperone, BiP. Prolonged ER stress induces the spliced leader RNA silencing (SLS) pathway. SLS is the trypanosome-specific stress response mechanism that elicits the shut-off of SL RNA transcription by perturbing the binding of the transcription factor tSNAP42 to its cognate promoter, eliminating trans-splicing of all mRNAs. SLS was discovered in the RNAi silenced cells depleted for functions that mediate translocation of proteins to the ER such as the signal recognition particle receptor SRα, SEC63- a factor that participates in protein translocation across the ER membrane, or SEC61- the translocation channel. Induction of SLS, either by prolonged ER stress or silencing of the genes associated with the ER membrane that function in ER protein translocation led to programmed cell death (PCD), evident by the exposure of phosphatidyl serine, DNA laddering, increase in ROS production, increase in cytoplasmic Ca(2+), and decrease in mitochondrial membrane potential. Here, we describe the protocols to induce ER stress and to observe the resulting morphological changes by transmission electron microscopy (TEM), changes in cytoplasmic Ca(2+), and DNA fragmentation which are the hallmarks of programmed cell death.

Role of protein translocation pathways across the endoplasmic reticulum in Trypanosoma brucei.

The translocation of secretory and membrane proteins across the endoplasmic reticulum (ER) membrane is mediated by co-translational (via the signal recognition particle (SRP)) and post-translational mechanisms. In this study, we investigated the relative contributions of these two pathways in trypanosomes. A homologue of SEC71, which functions in the post-translocation chaperone pathway in yeast, was identified and silenced by RNA interference. This factor is essential for parasite viability. In SEC71-silenced cells, signal peptide (SP)-containing proteins traversed the ER, but several were mislocalized, whereas polytopic membrane protein biogenesis was unaffected. Surprisingly trypanosomes can interchangeably utilize two of the pathways to translocate SP-containing proteins except for glycosylphosphatidylinositol-anchored proteins, whose level was reduced in SEC71-silenced cells but not in cells depleted for SRP68, an SRP-binding protein. Entry of SP-containing proteins to the ER was significantly blocked only in cells co-silenced for the two translocation pathways (SEC71 and SRP68). SEC63, a factor essential for both translocation pathways in yeast, was identified and silenced by RNA interference. SEC63 silencing affected entry to the ER of both SP-containing proteins and polytopic membrane proteins, suggesting that, as in yeast, this factor is essential for both translocation pathways in vivo. This study suggests that, unlike bacteria or other eukaryotes, trypanosomes are generally promiscuous in their choice of mechanism for translocating SP-containing proteins to the ER, although the SRP-independent pathway is favored for glycosylphosphatidylinositol-anchored proteins, which are the most abundant surface proteins in these parasites.

Spliced-leader RNA silencing: a novel stress-induced mechanism in Trypanosoma brucei.

The signal-recognition particle (SRP) mediates the translocation of membrane and secretory proteins across the endoplasmic reticulum upon interaction with the SRP receptor. In trypanosomes, the main RNA molecule is the spliced-leader (SL) RNA, which donates the SL sequence to all messenger RNA through trans-splicing. Here, we show that RNA interference silencing of the SRP receptor (SRalpha) in Trypanosoma brucei caused the accumulation of SRP on ribosomes and triggered silencing of SL RNA (SLS). SLS was elicited due to the failure of the SL RNA-specific transcription factor tSNAP42 to bind to its promoter. SL RNA reduction, in turn, eliminated mRNA processing and resulted in a significant reduction of all mRNA tested. SLS was also induced under pH stress and might function as a master regulator in trypanosomes. SLS is reminiscent of, but distinct from, the unfolded protein response and can potentially act as a new target for parasite eradication.

Down-regulation of the trypanosomatid signal recognition particle affects the biogenesis of polytopic membrane proteins but not of signal peptide-containing proteins.

Protein translocation across the endoplasmic reticulum is mediated by the signal recognition particle (SRP). In this study, the SRP pathway in trypanosomatids was down-regulated by two approaches: RNA interference (RNAi) silencing of genes encoding SRP proteins in Trypanosoma brucei and overexpression of dominant-negative mutants of 7SL RNA in Leptomonas collosoma. The biogenesis of both signal peptide-containing proteins and polytopic membrane proteins was examined using endogenous and green fluorescent protein-fused proteins. RNAi silencing of SRP54 or SRP68 in T. brucei resulted in reduced levels of polytopic membrane proteins, but no effect on the level of signal peptide-containing proteins was observed. When SRP deficiency was achieved in L. collosoma by overexpression of dominant-negative mutated 7SL RNA, a major effect was observed on polytopic membrane proteins but not on signal peptide-containing proteins. This study included two trypanosomatid species, tested various protein substrates, and induced depletion of the SRP pathway by affecting either the levels of SRP binding proteins or that of SRP RNA. Our results demonstrate that, as in bacteria but in contrast to mammalian cells, the trypanosome SRP is mostly essential for the biogenesis of membrane proteins.

The Trypanosoma brucei signal recognition particle lacks the Alu-domain-binding proteins: purification and functional analysis of its binding proteins by RNAi.

Trypanosomes are protozoan parasites that have a major impact on human health and that of livestock. These parasites represent a very early branch in the eukaryotic lineage, and possess unique RNA processing mechanisms. The trypanosome signal recognition particle (SRP) is also unusual in being the first signal recognition particle described in nature to be comprised of two RNA molecules, the 7SL RNA and a tRNA-like molecule. In this study, we further elucidated the unique properties of this particle. The genes encoding three SRP proteins (SRP19, SRP72 and SRP68) were identified by bioinformatics analysis. Silencing of these genes by RNAi suggests that the SRP-mediated protein translocation pathway is essential for growth. The depletion of SRP72 and SRP68 induced sudden death, most probably as a result of toxicity due to the accumulation of the pre-SRP in the nucleolus. Purification of the trypanosome particle to homogeneity, by TAP-tagging, identified four SRP proteins (SRP72, SRP68, SRP54 and SRP19), but no Alu-domain-binding protein homologs. This study highlights the unique features of the trypanosome SRP complex and further supports the hypothesis that the tRNA-like molecule present in this particle may replace the function of the Alu-domain-binding proteins present in many eukaryotic SRP complexes.