Longitudinal host transcriptional responses to SARS-CoV-2 infection in adults with extremely high viral load (preprint)

Current understanding of viral dynamics of SARS-CoV-2 and host responses driving the pathogenic mechanisms in COVID-19 is rapidly evolving. Here, we conducted a longitudinal study to investigate gene expression patterns during acute SARS-CoV-2 illness. Cases included SARS-CoV-2 infected individuals with extremely high viral loads early in their illness, individuals having low SARS-CoV-2 viral loads early in their infection, and individuals testing negative for SARS-CoV-2. We could identify widespread transcriptional host responses to SARS-CoV-2 infection that were initially most strongly manifested in patients with extremely high initial viral loads, then attenuating within the patient over time as viral loads decreased. Genes correlated with SARS-CoV-2 viral load over time were similarly differentially expressed across independent datasets of SARS-CoV-2 infected lung and upper airway cells, from both in vitro systems and patient samples. We also generated expression data on the human nose organoid model during SARS-CoV-2 infection. The human nose organoid-generated host transcriptional response captured many aspects of responses observed in the above patient samples while suggesting the existence of distinct host responses to SARS-CoV-2 depending on the cellular context, involving both epithelial and cellular immune responses. Our findings provide a catalog of SARS-CoV-2 host response genes changing over time.

The human nose organoid respiratory virus model: an ex-vivo human challenge model to study RSV and SARS-CoV-2 pathogenesis and evaluate therapeutics (preprint)

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.

Transmission event of SARS-CoV-2 Delta variant reveals multiple vaccine breakthrough infections (preprint)

Importance: Vaccine breakthrough by an emergent SARS-CoV-2 variant poses a great risk to global public health. Objective: To determine the SARS-CoV-2 variant responsible for 6 cases of vaccine breakthrough. Design: Nasopharyngeal 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. Setting: Transmission event occurred at events surrounding a wedding outside of Houston, TX. Two patients from India, likely transmitted the Delta variant to other guests. Participants: Following 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.

Oligonucleotide Capture Sequencing of the SARS-CoV-2 Genome and Subgenomic Fragments from COVID-19 Individuals (preprint)

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.

Evaluating recovery, cost, and throughput of different concentration methods for SARS-CoV-2 wastewater-based epidemiology (preprint)

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

Wastewater Analysis of SARS-CoV-2 as a Predictive Metric of Positivity Rate for a Major Metropolis (preprint)

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.

Identification of potential key genes for SARS-CoV-2 infected human bronchial organoids based on bioinformatics analysis (preprint)

There is an urgent need to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) that leads to COVID-19 and respiratory failure. Our study is to discover differentially expressed genes (DEGs) and biological signaling pathways by using a bioinformatics approach to elucidate their potential pathogenesis. The gene expression profiles of the GSE150819 datasets were originally produced using an Illumina NextSeq 500 (Homo sapiens). KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) were utilized to identify functional categories and significant pathways. KEGG and GO results suggested that the Cytokine-cytokine receptor interaction, P53 signaling pathway, and Apoptosis are the main signaling pathways in SARS-CoV-2 infected human bronchial organoids (hBOs). Furthermore, NFKBIA, C3, and CCL20 may be key genes in SARS-CoV-2 infected hBOs. Therefore, our study provides further insights into the therapy of COVID-19.

NVX-CoV2373 vaccine protects cynomolgus macaque upper and lower airways against SARS-CoV-2 challenge (preprint)

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

First-in-Human Trial of a SARS CoV 2 Recombinant Spike Protein Nanoparticle Vaccine (preprint)

Background NVX-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. Methods This 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. Results Participants 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. Conclusions NVX-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).

Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals (preprint)

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.

SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 elicits immunogenicity in baboons and protection in mice (preprint)

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).