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There is an unmet need for pre-clinical models to understand the pathogenesis of human respiratory viruses; and predict responsiveness to immunotherapies. Airway organoids can serve as an ex-vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a non-invasive technique to generate human nose organoids (HNOs) as an alternate to biopsy derived organoids. We made air liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hyper-secretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation) while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration dependent manner. Thus, the HNO-ALI model can serve as an alternate to lung organoids to study respiratory viruses and testing therapeutics.
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ImportanceVaccine breakthrough by an emergent SARS-CoV-2 variant poses a great risk to global public health. ObjectiveTo determine the SARS-CoV-2 variant responsible for 6 cases of vaccine breakthrough. DesignNasopharyngeal swabs from suspected vaccine breakthrough cases were tested for SARS-CoV-2 by qPCR for Wuhan-Hu1 and Alpha variant. Positive samples were then sequenced by Swift Normalase Amplicon Panels to determine the causal variant. SettingTransmission event occurred at events surrounding a wedding outside of Houston, TX. Two patients from India, likely transmitted the Delta variant to other guests. ParticipantsFollowing a positive SARS-CoV-2 qPCR test at a third-party site, six fully vaccinated patients were investigated. Three males and three females ranged from 53 to 69 years old. One patient suffered from diabetes while three others were classified as overweight. No significant other comorbidities were identified. None of the patients had a history of failed vaccination. Key PointsO_ST_ABSQuestionC_ST_ABSWhich SARS-CoV-2 variant is responsible for 6 cases of vaccine breakthrough, one interventional monoclonal antibody treatment, and one death? FindingsViral sequencing revealed 6 vaccinated patients were infected with the Delta SARS-CoV-2 variant. With no histories of vaccine breakthrough, this suggests Delta variant may possess immune evasion in patients that received the Pfizer BNT162b2, Moderna mRNA-1273, and Covaxin BBV152. MeaningDelta variant may pose the highest risk out of any currently circulating SARS-CoV-2 variants, with increased transmissibility over Alpha variant and possible vaccine breakthrough.
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The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity and provided evidence of expression of ORF10. Heterogeneous allelic frequencies along the 20kb ORF1ab gene suggested the presence of a defective interfering viral RNA species subpopulation in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.
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As the COVID-19 pandemic continues to affect communities across the globe, the need to contain the spread of the outbreaks is of paramount importance. Wastewater monitoring of the SARS-CoV-2 virus, the causative agent responsible for COVID-19, has emerged as a promising tool for health officials to anticipate outbreaks. As interest in wastewater monitoring continues to grow and municipalities begin to implement this approach, there is a need to further identify and evaluate methods used to concentrate SARS-CoV-2 virus RNA from wastewater samples. Here we evaluate the recovery, cost, and throughput of five different concentration methods for quantifying SARS-CoV-2 virus RNA in wastewater samples. We tested the five methods on six different wastewater samples. We also evaluated the use of a bovine coronavirus vaccine as a process control and pepper mild mottle virus as a normalization factor. Of the five methods we tested head-to-head, we found that HA filtration with bead beating performed the best in terms of sensitivity and cost. This evaluation can serve as a guide for laboratories establishing a protocol to perform wastewater monitoring of SARS-CoV-2. HighlightsO_LIFive methods for concentrating SARS-CoV-2 RNA from wastewater evaluated C_LIO_LIMethod performance characterized via recovery, cost, throughput, and variability C_LIO_LIHA filtration with bead beating had highest recovery for comparatively low cost C_LIO_LIBovine coronavirus, pepper mild mottle virus assessed as possible recovery controls C_LI
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Wastewater monitoring for SARS-CoV-2 has been suggested as an epidemiological indicator of community infection dynamics and disease prevalence. We report wastewater viral RNA levels of SARS-CoV-2 in a major metropolis serving over 3.6 million people geographically spread over 39 distinct sampling sites. Viral RNA levels were followed weekly for 22 weeks, both before, during, and after a major surge in cases, and simultaneously by two independent laboratories. We found SARS-CoV-2 RNA wastewater levels were a strong predictive indicator of trends in the nasal positivity rate two-weeks in advance. Furthermore, wastewater viral RNA loads demonstrated robust tracking of positivity rate for populations served by individual treatment plants, findings which were used in real-time to make public health interventions, including deployment of testing and education strike teams.
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BackgroundNVX-CoV2373 is a recombinant nanoparticle vaccine composed of trimeric full-length SARS-CoV-2 spike glycoproteins. We present the Day 35 primary analysis of our trial of NVX-CoV2373 with or without the saponin-based Matrix-M1 adjuvant in healthy adults. MethodsThis is a randomized, observer-blinded, placebo-controlled, phase 1 trial in 131 healthy adults. Trial vaccination comprised two intramuscular injections, 21 days apart. Primary outcomes were reactogenicity, safety labs, and immunoglobulin G (IgG) anti-spike protein response. Secondary outcomes included adverse events, wild-type virus neutralizing antibody, and T-cell responses. ResultsParticipants received NVX-CoV2373 with or without Matrix-M1 (n=106) or placebo (n=25). There were no serious adverse events. Reactogenicity was mainly mild in severity and of short duration (mean [≤]2 days), with second vaccinations inducing greater local and systemic reactogenicity. The adjuvant significantly enhanced immune responses and was antigen dose-sparing, and the two-dose 5g NVX-CoV2373/Matrix-M1 vaccine induced mean anti-spike IgG and neutralizing antibody responses that exceeded the mean responses in convalescent sera from COVID-19 patients with clinically significant illnesses. The vaccine also induced antigen-specific T cells with a largely T helper 1 (Th1) phenotype. ConclusionsNVX-CoV2373/Matrix-M1 was well tolerated and elicited robust immune responses (IgG and neutralization) four-fold higher than the mean observed in COVID-19 convalescent serum from participants with clinical symptoms requiring medical care and induced CD4+ T-cell responses biased toward a Th1 phenotype. These findings suggest that the vaccine may confer protection and support transition to efficacy evaluations to test this hypothesis. (Funded by the Coalition for Epidemic Preparedness Innovations; ClinicalTrials.gov number, NCT04368988).
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There is an urgent need for a safe and protective vaccine to control the global spread of SARS-CoV-2 and prevent COVID-19. Here, we report the immunogenicity and protective efficacy of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) produced from the full-length SARS-CoV-2 spike (S) glycoprotein stabilized in the prefusion conformation. Cynomolgus macaques (Macaca fascicularis) immunized with NVX-CoV2373 and the saponin-based Matrix-M adjuvant induced anti-S antibody that was neutralizing and blocked binding to the human angiotensin-converting enzyme 2 (hACE2) receptor. Following intranasal and intratracheal challenge with SARS-CoV-2, immunized macaques were protected against upper and lower infection and pulmonary disease. These results support ongoing phase 1/2 clinical studies of the safety and immunogenicity of NVX-CoV2327 vaccine (NCT04368988). HighlightsO_LIFull-length SARS-CoV-2 prefusion spike with Matrix-M1 (NVX-CoV2373) vaccine. C_LIO_LIInduced hACE2 receptor blocking and neutralizing antibodies in macaques. C_LIO_LIVaccine protected against SARS-CoV-2 replication in the nose and lungs. C_LIO_LIAbsence of pulmonary pathology in NVX-CoV2373 vaccinated macaques. C_LI
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The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity and provided evidence of expression of ORF10. Heterogeneous allelic frequencies along the 20kb ORF1ab gene suggested the presence of a defective interfering viral RNA species subpopulation in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.
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The COVID-19 pandemic continues to spread throughout the world with an urgent need for a safe and protective vaccine to effectuate herd immunity to control the spread of SARS-CoV-2. Here, we report the development of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) produced from the full-length spike (S) protein, stabilized in the prefusion conformation. Purified NVX-CoV2373 S form 27.2nm nanoparticles that are thermostable and bind with high affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. In mice and baboons, low-dose NVX-CoV2373 with saponin-based Matrix-M adjuvant elicits high titer anti-S IgG that is associated with blockade of hACE2 receptor binding, virus neutralization, and protection against SARS-CoV-2 challenge in mice with no evidence of vaccine-associated enhanced respiratory disease (VAERD). NVX-CoV2373 vaccine also elicits multifunctional CD4+ and CD8+ T cells, CD4+ T follicular helper T cells (Tfh), and the generation of antigen-specific germinal center (GC) B cells in the spleen. These results support the ongoing phase 1/2 clinical evaluation of the safety and immunogenicity of NVX-CoV2327 with Matrix-M (NCT04368988).
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PURPOSE: The purpose of this study was to document bronchiolitis obliterans, the long term pulmonary sequelae of adenovirus type 7 pneumonia, using High Resolution CT(HRCT) and to identify risk factors that increase the likelihood of developing bronchiolitis obliterans. METHODS: Thirty-nine children were admitted to the Inje University, Sanggye Paik Hospital in the early summer of 1996 with clinical adenoviral pnemonia. Type 7 adenovirus was isolated from 11 children. All eleven were reassessed clinically and performed HRCT in 1997, one year (12.1+/-1.9 mo) after the adenovirus type 7 pnemonia. RESULTS: Seven (64%) had HRCT findings of BO(BO group) including mosaic perfusion, attenuation of pulmonary vessel and bronchiectasis, Four(36%) had a normal HRCT finding(Non-BO group). The mean age of the children on admission was lower in BO group(15.3+/-7.1 mo) than Non-BO group(35.3+/-14.2 mo) (P<0.05). The mean duration of fever was longer in BO group(14.4+/-2.8 days) than Non-BO group(6.3+/-2.5 days)(P<0.01). The mean duration of initial hospital admission was longer in BO group(28.0+/-10.8 days) than Non-BO group (9.8+/-3.6 days)(P<0.01). But the sex ratio between the two groups was not statistically significant. Four of the seven BO group subjects had bronchial asthma, and three of the four patients had persistent wheezing . CONCLUSIONS: Adenovirus type 7 pneumonia is closely associated with bronchiolitis obliterans, the important long term pulmonary sequelae. The risk factors which increase the possibility of developing bronchiolitis obliterans are: the young age of the child, the long fever duration , and the long admission duration.
