The variable conversion of neutralizing anti-SARS-CoV-2 single-chain antibodies to IgG provides insight into RBD epitope accessibility.

Monoclonal antibody (mAb) therapies have rapidly become a powerful class of therapeutics with applications covering a diverse range of clinical indications. Though most widely used for the treatment of cancer, mAbs are also playing an increasing role in the defense of viral infections, most recently with palivizumab for prevention and treatment of severe RSV infections in neonatal and pediatric populations. In addition, during the COVID-19 pandemic, mAbs provided a bridge to the rollout of vaccines; however, their continued role as a therapeutic option for those at greatest risk of severe disease has become limited due to the emergence of neutralization resistant Omicron variants. Although there are many techniques for the identification of mAbs, including single B cell cloning and immunization of genetically engineered mice, the low cost, rapid throughput and technological simplicity of antibody phage display has led to its widespread adoption in mAb discovery efforts. Here we used our 27-billion-member naïve single-chain antibody (scFv) phage library to identify a panel of neutralizing anti-SARS-CoV-2 scFvs targeting diverse epitopes on the receptor binding domain (RBD). Although typically a routine process, we found that upon conversion to IgG, a number of our most potent clones failed to maintain their neutralization potency. Kinetic measurements confirmed similar affinity to the RBD; however, mechanistic studies provide evidence that the loss of neutralization is a result of structural limitations likely arising from initial choice of panning antigen. Thus this work highlights a risk of scFv-phage panning to mAb conversion and the importance of initial antigen selection.

Mycobacterium tuberculosis grows linearly at the single-cell level with larger variability than model organisms.

The ability of bacterial pathogens to regulate growth is crucial to control homeostasis, virulence, and drug response. Yet, we do not understand the growth and cell cycle behaviors of Mycobacterium tuberculosis (Mtb), a slow-growing pathogen, at the single-cell level. Here, we use time-lapse imaging and mathematical modeling to characterize these fundamental properties of Mtb. Whereas most organisms grow exponentially at the single-cell level, we find that Mtb exhibits a unique linear growth mode. Mtb growth characteristics are highly variable from cell-to-cell, notably in their growth speeds, cell cycle timing, and cell sizes. Together, our study demonstrates that growth behavior of Mtb diverges from what we have learned from model bacteria. Instead, Mtb generates a heterogeneous population while growing slowly and linearly. Our study provides a new level of detail into how Mtb grows and creates heterogeneity, and motivates more studies of growth behaviors in bacterial pathogens.

IgG-like bispecific antibodies with potent and synergistic neutralization against circulating SARS-CoV-2 variants of concern.

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.

Unique structural solution from a VH3-30 antibody targeting the hemagglutinin stem of influenza A viruses.

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.

Ultra-Sensitive Serial Profiling of SARS-CoV-2 Antigens and Antibodies in Plasma to Understand Disease Progression in COVID-19 Patients with Severe Disease.

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 patients with coronavirus disease (COVID-19). We studied plasma from 64 patients who were COVID-19 positive, 17 who were COVID-19 negative, and 34 prepandemic 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 patients with COVID-19. 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 1 day) was statistically significant. CONCLUSIONS: The reported SARS-CoV-2 Simoa antigen assay is the first to detect viral antigens in the plasma of patients who were COVID-19 positive 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.

Flavors of Flaviviral RNA Structure: towards an Integrated View of RNA Function from Translation through Encapsidation.

For many viruses, RNA is the holder of genetic information and serves as the template for both replication and translation. While host and viral proteins play important roles in viral decision-making, the extent to which viral RNA (vRNA) actively participates in translation and replication might be surprising. Here, the focus is on flaviviruses, which include common human scourges such as dengue, West Nile, and Zika viruses, from an RNA-centric viewpoint. In reviewing more recent findings, an attempt is made to fill knowledge gaps and revisit some canonical views of vRNA structures involved in replication. In particular, alternative views are offered on the nature of the flaviviral promoter and genome cyclization, and the feasibility of refining in vitro-derived models with modern RNA probing and sequencing methods is pointed out. By tracing vRNA structures from translation through encapsidation, a dynamic molecule closely involved in the self-regulation of viral replication is revealed.

Identification of a Conserved RNA-dependent RNA Polymerase (RdRp)-RNA Interface Required for Flaviviral Replication.

Dengue virus, an ∼10.7-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.

Characterization of soluble RNA-dependent RNA polymerase from dengue virus serotype 2: The polyhistidine tag compromises the polymerase activity.

The viral RNA polymerase is an attractive target for inhibition in the treatment of viral infections. In the case of dengue virus (DENV), a member of the genus Flavivirus, the RNA-dependent RNA polymerase (RdRp) activity resides in the C-terminal two-thirds of non-structural protein (NS) 5 responsible for the de novo synthesis of the viral RNA genome. Among four distinct, but closely related dengue serotypes, serotype 2 (DENV-2) produces more severe diseases than other serotypes. It has been reported that bacterial production of the recombinant DENV-2 RdRp was difficult due to its low expression and solubility levels. To facilitate functional and structural analyses, we here demonstrate complete protocols for overexpression and purification of soluble DENV-2 RdRp, increasing protein yields by a remarkable 10 times compared to earlier reports. Three different forms of DENV-2 RdRp as either N- or C-terminally His-tagged fusions, or without tag, were purified to homogeneity. We show here that the presence of both the N- and C-terminal His-tag had a deleterious effect on polymerase activity and, in contrast to earlier studies, our non-tagged RdRp did not require manganese ions to activate RNA polymerization. We also determined an apparent Kd value of 53nM for binding to the 5'-UTR RNA by surface plasmon resonance (SPR). Our work provide a more suitable material for basic research of viral RdRp and for drug development.