Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 21 million people worldwide since August 16, 2020. Compared to PCR and serology tests, SARS-CoV-2 antigen assays are underdeveloped, despite their potential to identify active infection and monitor disease progression. Methods We used Single Molecule Array (Simoa) assays to quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of coronavirus disease (COVID-19) patients. We studied plasma from 64 COVID-19 positive patients, 17 COVID-19 negative patients, and 34 pre-pandemic patients. Combined with Simoa anti-SARS-CoV-2 serological assays, we quantified changes in 31 SARS-CoV-2 biomarkers in 272 longitudinal plasma samples obtained for 39 COVID-19 patients. Data were analyzed by hierarchical clustering and were compared to longitudinal RT-PCR test results and clinical outcomes. Results SARS-CoV-2 S1 and N antigens were detectable in 41 out of 64 COVID-19 positive patients. In these patients, full antigen clearance in plasma was observed a mean ± 95%CI of 5 ± 1 days after seroconversion and nasopharyngeal RT-PCR tests reported positive results for 15 ± 5 days after viral antigen clearance. Correlation between patients with high concentrations of S1 antigen and ICU admission (77%) and time to intubation (within one day) was statistically significant. Conclusions The reported SARS-CoV-2 Simoa antigen assay is the first to detect viral antigens in the plasma of COVID-19 positive patients to date. These data show that SARS-CoV-2 viral antigens in the blood are associated with disease progression, such as respiratory failure, in COVID-19 cases with severe disease.
Dengue virus, an ϳ10.7-kb positive-sense RNA virus, is the most common arthropod-communicated pathogen in the world. Despite dengue's clear epidemiological importance, mechanisms for its replication remain elusive. Here, we probed the entire dengue genome for interactions with viral RNA-dependent RNA polymerase (RdRp), and we identified the dominant interaction as a loop-forming ACAG motif in the 3 positive-stranded terminus, complicating the prevailing model of replication. A subset of interactions coincides with known flaviviral recombination sites inside the viral protein-coding region. Specific recognition of the RNA element occurs via an arginine patch in the C-terminal thumb domain of RdRp. We also show that the highly conserved nature of the consensus RNA motif may relate to its tolerance to various mutations in the interacting region of RdRp. Disruption of the interaction resulted in loss of viral replication ability in cells. This unique RdRp-RNA interface is found throughout flaviviruses, implying possibilities for broad disease interventions.Positive-sense single-stranded RNA viruses initiate replication by the generation of a complementary negative (Ϫ) RNA strand via the action of viral RNA-dependent RNA polymerases (RdRps). 3 In the cases of brome mosaic virus (family Bromoviridae), turnip crinkle virus (Tombusviridae), hepatitis C virus (Flaviviridae), and encephalomyocarditis virus (Picornaviridae), RNA structures in the 3Ј-untranslated region (UTR) of the positive strand have been characterized as RdRp promoters (1-4). In the case of dengue virus (DENV), a flavivirus with a 5Ј-type I cap and the absence of a polyadenylated tail, however, the current model for replication asserts that the 5Ј-UTR acts as a promoter for (Ϫ)-strand synthesis. This position is supported by several lines of in vitro evidence. In particular, atomic force microscopy demonstrated cyclization of the (ϩ)-strand genomic RNA, resulting in placement of the 5Ј-UTR in proximity to the 3Ј terminus, whereas EMSA and footprinting assays apparently documented RdRp interactions with the first hairpin element in the DENV 5Ј-UTR, designated stem-loop A (SLA) (5). In addition, an RNA fragment of the DENV 5Ј-UTR could also stimulate in vitro synthesis of a complementary product of the 3Ј-UTR (5, 6).To date, investigations of RdRp actions on the flaviviral RNA genome have been limited to in vitro assays on less than 5% of the whole genome with a bias toward UTR regions; therefore, the entire interaction landscape of RdRp has not yet been revealed. It is also unclear whether flaviviral RdRps require a specific RNA promoter for de novo initiation of RNA synthesis. To gain insight into accurate mechanisms for synthesis of the viral genome in the cell, here we explored all possible interactions between RdRp and the complete RNA genome in an unbiased in vivo context using a refined yeast three-hybrid (Y3H) scan. Combining bioinformatic evidence with in vitro binding and viral replicon assays, we pinpoint amino acids and nucleotides contributin...
Broadly neutralizing antibodies (bnAbs) targeting conserved influenza A virus (IAV) hemagglutinin (HA) epitopes can provide valuable information for accelerating universal vaccine designs. Here, we report structural details for heterosubtypic recognition of HA from circulating and emerging IAVs by the human antibody 3I14. Somatic hypermutations play a critical role in shaping the HCDR3, which alone and uniquely among VH3-30 derived antibodies, forms contacts with five sub-pockets within the HA-stem hydrophobic groove. 3I14 light-chain interactions are also key for binding HA and contribute a large buried surface area spanning two HA protomers. Comparison of 3I14 to bnAbs from several defined classes provide insights to the bias selection of VH3-30 antibodies and reveals that 3I14 represents a novel structural solution within the VH3-30 repertoire. The structures reported here improve our understanding of cross-group heterosubtypic binding activity, providing the basis for advancing immunogen designs aimed at eliciting a broadly protective response to IAV.
Monoclonal antibodies are a promising approach to treat COVID-19, however the emergence of SARS-CoV-2 variants has challenged the efficacy and future of these therapies. Antibody cocktails are being employed to mitigate these challenges, but neutralization escape remains a major challenge and alternative strategies are needed. Here we present two anti-SARS-CoV-2 spike binding antibodies, one Class 1 and one Class 4, selected from our non-immune human single-chain variable fragment (scFv) phage library, that are engineered into four, fully-human IgG-like bispecific antibodies (BsAb). Prophylaxis of hACE2 mice and post-infection treatment of golden hamsters demonstrates the efficacy of the monospecific antibodies against the original Wuhan strain, while promising in vitro results with the BsAbs demonstrate enhanced binding and distinct synergistic effects on neutralizing activity against circulating variants of concern. In particular, one BsAb engineered in a tandem scFv-Fc configuration shows synergistic neutralization activity against several variants of concern including B.1.617.2. This work provides evidence that synergistic neutralization can be achieved using a BsAb scaffold, and serves as a foundation for the future development of broadly reactive BsAbs against emerging variants of concern.
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