SARS-CoV-2 Omicron is specifically restricted in its replication in human lung tissue, compared to other variants of concern

SARS-CoV-2 Omicron variant has been characterized by decreased clinical severity, raising the question of whether early variant-specific interactions within the mucosal surfaces of the respiratory tract could mediate its attenuated pathogenicity. Here, we employed ex vivo infection of native human nasal and lung tissues to investigate the local-mucosal susceptibility and innate immune response to Omicron, compared to Delta and earlier SARS-CoV-2 variants of concern (VOC). We show that the replication of Omicron in lung tissues is highly restricted compared to other VOC, whereas it remains relatively unchanged in nasal tissues. Mechanistically, Omicron induced a much stronger antiviral interferon response in infected tissues compared to Delta and earlier VOC - a difference which was most striking in the lung tissues, where the innate immune response to all other SARS-CoV-2 VOC was blunted. Our data provide new insights to the reduced lung involvement and clinical severity of Omicron.

Intramuscular mRNA BNT162b2 vaccine against SARS-CoV-2 induces robust neutralizing salivary IgA

Intramuscularly administered vaccines stimulate robust serum neutralizing antibodies, yet they are often less competent in eliciting sustainable sterilizing immunity at the mucosal level. Our study uncovers, strong neutralizing mucosal component (NT50 [≤] 50pM), emanating from intramuscular administration of an mRNA vaccine. We show that saliva of BNT162b2 vaccinees contains temporary IgA targeting the Receptor-Binding-Domain (RBD) of SARS-CoV-2 spike protein and demonstrate that these IgAs are key mediators of potent neutralization. RBD-targeting IgAs were found to associate with the Secretory Component, indicating their bona-fide transcytotic origin and their dimeric tetravalent nature. The mechanistic understanding of the exceptionally high neutralizing activity provided by mucosal IgA, acting at the first line of defence, will advance vaccination design and surveillance principles, pointing to novel treatment approaches, and to new routes of vaccine administration and boosting. Significance statementWe unveiled powerful mucosal neutralization upon BNT162b2 vaccination, mediated by temporary polymeric IgA and explored its longitudinal properties. We present a model, whereby the molecular architecture of polymeric mucosal IgA and its spatial properties are responsible for the outstanding SARS-CoV-2 neutralization potential. We established a methodology for quantitative comparison of immunoreactivity and neutralization for IgG and IgAs in serum and saliva in molar equivalents for standardization in diagnostics, surveillance of protection and for vaccine evaluations.

Early versus late third trimester maternal SARS-CoV-2 BNT162b2 mRNA immunization maximizes transplacental antibody transfer and neonatal neutralizing antibody levels

ObjectiveWe aimed to assess the impact of early versus late third trimester maternal SARS-CoV-2 vaccination on transplacental transfer and neonatal levels of SARS-CoV-2 antibodies. MethodsMaternal and cord blood sera were collected following term delivery after antenatal SARS-CoV-2 BNT162b2 mRNA vaccination, with the first vaccine dose administered during 27-36 weeks gestation. SARS-CoV-2 spike protein (S) and receptor binding domain (RBD)- specific, IgG levels and neutralizing potency were evaluated in maternal and cord blood samples. ResultsThe study cohort consisted of 171 parturients (median age, 31 years; median gestational age, 39.7 weeks): 83 (48.5%) immunized at early 3rd trimester (1st dose at 27-31 weeks), and 88 (51.5%) immunized at late 3rd trimester (1st dose at 32-36 weeks). All mother-infant paired sera were positive for anti S- and anti-RBD-specific IgG. Anti-RBD-specific IgG concentrations in neonatal sera were higher following early versus late 3rd trimester vaccination and were positively correlated with increasing time since vaccination (r={square}0.26; P=0.001). The median placental transfer ratios of anti-S and anti-RBD specific IgG were increased following early versus late 3rd trimester immunization (anti-S ratio:1.3 vs. 0.9, anti-RBD-specific ratio:2.3 vs. 0.7, P<0.001). Neutralizing antibodies placental transfer ratio was greater following early versus late 3rd trimester immunization (1.9 vs. 0.8, P<0.001), and was positively associated with longer duration from vaccination (r={square}0.77; P<0.001). ConclusionsEarly- as compared to late third trimester maternal SARS-CoV-2 immunization enhanced transplacental antibody transfer and increased neonatal neutralizing antibody levels. Our findings highlight that vaccination of pregnant women early in the third trimester may optimize neonatal seroprotection.

The 5-months impact of tozinameran (BNT162b2) mRNA vaccine on kidney transplant and chronic dialysis patients

BackgroundDetermining the humoral immunogenicity of tozinameran (BNT162b2) vaccine in patients requiring chronic renal replacement therapy, and its impact on COVID-19 morbidity several months after vaccination, will guide risk assessment and subsequent changes in vaccination policy. MethodsIn a prospective post-vaccination cohort study with up to 5 months follow-up we studied outpatient dialysis and kidney transplant patients and respective healthcare teams. Outcomes were anti S1/S2 antibody response to vaccine or infection and infection rate during followup. Results175 dialysis patients (40% women, 65{+/-}15 years), 252 kidney transplant patients (33% women, 54{+/-}14 years) and 71 controls (65% women, 44{+/-}14 years) were followed. Three months or longer after vaccination we detected anti S1/S2 IgG antibodies in 80% of dialysis patients, 44% of transplant recipients and 100% of controls, whereas respective rates after infection were 94%, 75% and 100%. Predictors of non-response were older age, diabetes, history of cancer, lower lymphocyte count and lower vitamin-D levels. Factors associated with lower titers in dialysis patients were modality (hemodialysis vs peritoneal) and high serum ferritin levels. In transplant patients, hypertension and higher calcineurin or mTOR inhibitor drug levels were linked with diminished antibody response. Vaccination associated with fewer subsequent infections (HR=0.23, p<0.05). Moreover, higher antibody titers associated with fewer events, HR 0.41 for each unit increased in log10titer (p<0.05). ConclusionsDialysis patients, and more so kidney transplant recipients, mounted reduced antibody response to COVID-19 mRNA vaccination, and lesser humoral response associated with more infections. Measures to identify and protect non-responsive patients are urgently required. SignificanceReports on the humoral immunogenicity of SARS-CoV-2 mRNA vaccines in patients with end stage renal disease are scarce, and association with subsequent COVID-19 morbidity is unknown. In this cohort study that included 175 patients treated with dialysis, 252 kidney transplant recipients and 71 control volunteers, the proportion achieving an antibody response was time- and group-dependent, reaching 80%, 44% and 100% at 3 months post prime inoculation. Personal history of vaccination, positive antibody responses and antibody titers associated with significantly lower risk of COVID-19 infection. Thus, in patients with end stage renal disease, SARS-CoV-2 antibody testing may be warranted after vaccination, to identify non-responders at higher risk for disease.

Ovarian follicular function is not altered by SARS-Cov-2 infection or BNT162b2 mRNA Covid-19 vaccination.

ImportanceThis is the first study to examine the impact of SARS-Cov-2 infection and COVID-19 vaccination on ovarian function. ObjectiveTo characterize anti-COVID-19 antibodies in follicular fluid and compare ovarian follicle function in women following confirmed SARS-CoV-2 infection, COVID-19 vaccination, and non-infected, unvaccinated controls. DesignThis is a cohort study conducted between February 1 and March 10, 2021. SettingA single university hospital-based IVF clinic. ParticipantsConsecutive sample of female patients undergoing oocyte retrieval. InterventionsConsenting patients were recruited and assigned to one of three study groups: recovering from confirmed COVID 19 (n=9); vaccinated (n=9); and uninfected, non-vaccinated controls (n=14). Serum and follicular fluid samples were taken and analyzed for anti-COVID IgG as well as estrogen, progesterone and HSPG2 concentration, as well as the number and maturity of aspirated oocytes and previous estrogen and progesterone measurements. Main outcome measuresFollicular function, including steroidogenesis, follicular response to the LH/hCG trigger, and oocyte quality biomarkers. ResultsBoth natural and vaccine elicited anti-COVID IgG antibodies were detected in the follicular fluid in levels proportional to the IgG serum concentration. No differences were detected in any of the surrogate ovarian follicle quality reporting parameters. Conclusions and relevanceBoth SARS-COV-2 infection and vaccination with the BNT162b2 mRNA vaccine mediate IgG immunity that crosses into the follicular fluid. No detrimental effect on follicular function was detected. Trial RegistrationCinicalTrials.gov registry number NCT04822012 Key PointCOVID 19 disease and BNT162b2 mRNA vaccine induce anti-COVID IgG in follicular fluid; neither recent infection nor vaccination appear to negatively effect follicular function.

Human nasal and lung tissues infected ex vivo with SARS-CoV-2 provide insights into differential tissue-specific and virus-specific innate immune responses in the upper and lower respiratory tract

The nasal-mucosa constitutes the primary entry site for respiratory viruses including SARS-CoV-2. While the imbalanced innate immune response of end-stage COVID-19 has been extensively studied, the earliest stages of SARS-CoV-2 infection at the mucosal entry site have remained unexplored. Here we employed SARS-CoV-2 and influenza virus infection in native multi-cell-type human nasal turbinate and lung tissues ex vivo, coupled with genome-wide transcriptional analysis, to investigate viral susceptibility and early patterns of local-mucosal innate immune response in the authentic milieu of the human respiratory tract. SARS-CoV-2 productively infected the nasal turbinate tissues, predominantly targeting respiratory epithelial cells, with rapid increase in tissue-associated viral sub-genomic mRNA, and secretion of infectious viral progeny. Importantly, SARS-CoV-2 infection triggered robust antiviral and inflammatory innate immune responses in the nasal mucosa. The upregulation of interferon stimulated genes, cytokines and chemokines, related to interferon signaling and immune-cell activation pathways, was broader than that triggered by influenza virus infection. Conversely, lung tissues exhibited a restricted innate immune response to SARS-CoV-2, with a conspicuous lack of type I and III interferon upregulation, contrasting with their vigorous innate immune response to influenza virus. Our findings reveal differential tissue-specific innate immune responses in the upper and lower respiratory tract, that are distinct to SARS-CoV-2. The studies shed light on the role of the nasal-mucosa in active viral transmission and immune defense, implying a window of opportunity for early interventions, whereas the restricted innate immune response in early-SARS-CoV-2-infected lung tissues could underlie the unique uncontrolled late-phase lung damage of advanced COVID-19. IMPORTANCEIn order to reduce the late-phase morbidity and mortality of COVID-19, there is a need to better understand and target the earliest stages of SARS-CoV-2 infection in the human respiratory tract. Here we have studied the initial steps of SARS-CoV-2 infection and the consequent innate immune responses within the natural multicellular complexity of human nasal-mucosal and lung tissues. Comparing the global innate response patterns of nasal and lung tissues, infected in parallel with SARS-CoV-2 and influenza virus, we have revealed distinct virus-host interactions in the upper and lower respiratory tract, which could determine the outcome and unique pathogenesis of SARS-CoV-2 infection. Studies in the nasal-mucosal infection model can be employed to assess the impact of viral evolutionary changes, and evaluate new therapeutic and preventive measures against SARS-CoV-2 and other human respiratory pathogens.

Efficient maternofetal transplacental transfer of anti- SARS-CoV-2 spike antibodies after antenatal SARS-CoV-2 BNT162b2 mRNA vaccination

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during pregnancy and early infancy can result in severe disease. Evaluating the serologic response after maternal vaccination during pregnancy and subsequent transplacental antibody transfer has important implications for maternal care and vaccination strategies. ObjectiveTo assess maternal and neonatal SARS-CoV-2 antibody levels after antenatal mRNA vaccination. Design, Setting, and ParticipantsThis study took place at Hadassah Medical Center in Jerusalem, Israel in February 2021. Maternal and cord blood sera were collected for antibody measurement from mother/newborn dyads following antenatal vaccination. ExposureSARS-CoV-2 BNT162b2 mRNA vaccination. Main outcome and measuresSpike protein (S) and receptor binding domain (RBD) - specific, IgG levels were evaluated in maternal and cord blood sera. ResultsThe study cohort consisted of 20 parturients, with a median maternal age of 32 y ears and a median gestational age of 393/7 weeks at the time of delivery. The median time lapsed from the first and second doses of vaccine administration until delivery was 33 [IQR 30-37] and 11 [IQR 9-15] days, respectively. Of the 20 dyads, all women an d infants were positive for anti S- and anti-RBD-specific IgG. Anti-S and anti-RBD-specific IgG levels in maternal sera were positively correlated to their respective concentrations in cord blood ({rho}s= 0.72; P<0.001 and {rho}s= 0.72; P <0.001, respectively). Anti-S and anti-RBD-specific IgG titers in cord blood were directly correlated with time lapsed since the administration of the first vaccine dose ({rho}s= 0.71; P =0.001 and {rho}s= 0.63; P=0.004, respectively). Conclusion and RelevanceIn this study, SARS-CoV-2 mRNA vaccine administered during pregnancy induced adequate maternal serologic response with subsequent efficient transplacental transfer. Our findings highlight that vaccination of pregnant women may provide maternal and neonatal protection from SARS-CoV-2 infection.

Lessons from applied large-scale pooling of 133,816 SARS-CoV-2 RT-PCR tests

Pooling multiple swab samples prior to RNA extraction and RT-PCR analysis was proposed as a strategy to reduce costs and increase throughput of SARS-CoV-2 tests. However, reports on practical large-scale group testing for SARS-CoV-2 have been scant. Key open questions concern reduced sensitivity due to sample dilution; the rate of false positives; the actual efficiency (number of tests saved by pooling) and the impact of infection rate in the population on assay performance. Here we report analysis of 133,816 samples collected at April-September 2020, tested by pooling for the presence of SARS-CoV-2. We spared 76% of RNA extraction and RT-PCR tests, despite the reality of frequently changing prevalence rate (0.5%-6%). Surprisingly, we observed pooling efficiency and sensitivity that exceed theoretical predictions, which resulted from non-random distribution of positive samples in pools. Overall, the findings strongly support the use of pooling for efficient large high throughput SARS-CoV-2 testing.

Improved Sensitivity, Safety, and Rapidity of COVID-19 Tests by Replacing Viral Storage Solution with Lysis Buffer

Conducting numerous, rapid, and reliable PCR tests for SARS-CoV-2 is essential for our ability to monitor and control the current COVID-19 pandemic. Here, we tested the sensitivity and efficiency of SARS-CoV-2 detection in clinical samples collected directly into a mix of lysis buffer and RNA preservative, thus inactivating the virus immediately after sampling. We tested 79 COVID-19 patients and 20 healthy controls. We collected two samples (nasopharyngeal swabs) from each participant: one swab was inserted into a test tube with Viral Transport Medium (VTM), following the standard guideline used as the recommended method for sample collection; the other swab was inserted into a lysis buffer supplemented with nucleic acid stabilization mix (coined NSLB). We found that RT-qPCR tests of patients were significantly more sensitive with NSLB sampling, reaching detection threshold 2.1{+/-}0.6 (Mean{+/-}SE) PCR cycles earlier then VTM samples from the same patient. We show that this improvement is most likely since NSLB samples are not diluted in lysis buffer before RNA extraction. Re-extracting RNA from NSLB samples after 72 hours at room temperature did not affect the sensitivity of detection, demonstrating that NSLB allows for long periods of sample preservation without special cooling equipment. We also show that swirling the swab in NSLB and discarding it did not reduce sensitivity compared to retaining the swab in the tube, thus allowing improved automation of COVID-19 tests. Overall, we show that using NSLB instead of VTM can improve the sensitivity, safety, and rapidity of COVID-19 tests at a time most needed.

ApharSeq: An Extraction-free Early-Pooling Protocol for Massively Multiplexed SARS-CoV-2 Detection

The global SARS-CoV-2 pandemic created a dire need for viral detection tests worldwide. Most current tests for SARS-CoV-2 are based on RNA extraction followed by quantitative reverse-transcription PCR assays. While automation and improved logistics increased the capacity of these tests, they cannot exceed the lower bound dictated by one extraction and one RT-PCR reaction per sample. Multiplexed next generation sequencing (NGS) assays provide a dramatic increase in throughput, and hold the promise of richer information including viral strains, host immune response, and multiple pathogens. Here, we establish a significant improvement of existing RNA-seq detection protocols. Our workflow, ApharSeq, includes a fast and cheap RNA capture step, that is coupled to barcoding of individual samples, followed by sample-pooling prior to the reverse transcription, PCR and massively parallel sequencing. Thus, only one non-enzymatic step is performed before pooling hundreds of barcoded samples for subsequent steps and further analysis. We characterize the quantitative aspects of the assay by applying ApharSeq to more than 500 clinical samples in a robotic workflow. The assay results are linear, and the empirical limit of detection is found to be Ct 33 (roughly 1000 copies/ml). A single ApharSeq test currently costs under 1.2$, and we estimate costs can further go down 3-10 fold. Similarly, we estimate a labor reduction of 10-100 fold, automated liquid handling of 5-10 fold, and reagent requirement reduction of 20-1000 fold compared to existing testing methods.

Pooled RNA extraction and PCR assay for efficient SARS-CoV-2 detection

Testing for active SARS-CoV-2 infection is a fundamental tool in the public health measures taken to control the COVID-19 pandemic. Due to the overwhelming use of SARS-CoV-2 RT-PCR tests worldwide, availability of test kits has become a major bottleneck. Here we demonstrate pooling strategies to perform RNA extraction and RT-PCR in pools, significantly increasing throughput while maintaining clinical sensitivity. We implemented the method in a routine clinical diagnosis setting of asymptomatic populations, and already tested 5,464 individuals for SARS-CoV-2 using 731 RNA extraction and RT-PCR kits. We identified six SARS-CoV-2 positive patients corresponding to 0.11% of the tested population.