SARS-CoV-2 spike glycosylation affects function and neutralization sensitivity (preprint)

The glycosylation of viral envelope proteins can play important roles in virus biology and immune evasion. The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) includes 22 N-linked glycosylation sequons and 17 O-linked glycosites. Here, we investigated the effect of individual glycosylation sites on SARS-CoV-2 S function in pseudotyped virus infection assays and on sensitivity to monoclonal and polyclonal neutralizing antibodies. In most cases, removal of individual glycosylation sites decreased the infectiousness of the pseudotyped virus. For glycosylation mutants in the N-terminal domain (NTD) and the receptor binding domain (RBD), reduction in pseudotype infectivity was predicted by a commensurate reduction in the level of virion-incorporated spike protein. Notably, the presence of a glycan at position N343 within the RBD had diverse effects on neutralization by RBD-specific monoclonal antibodies (mAbs) cloned from convalescent individuals. The N343 glycan reduced overall sensitivity to polyclonal antibodies in plasma from COVID-19 convalescent individuals, suggesting a role for SARS-CoV-2 spike glycosylation in immune evasion. However, vaccination of convalescent individuals produced neutralizing activity that was resilient to the inhibitory effect of the N343 glycan.

Molecular fate-mapping of serum antibodies reveals the effects of antigenic imprinting on repeated immunization (preprint)

The ability of serum antibody to protect against pathogens arises from the interplay of antigen-specific B cell clones of different affinities and fine specificities. These cellular dynamics are ultimately responsible for serum-level phenomena such as antibody imprinting or "Original Antigenic Sin" (OAS), a proposed propensity of the immune system to rely repeatedly on the first cohort of B cells that responded to a stimulus upon exposure to related antigens. Imprinting/OAS is thought to pose a barrier to vaccination against rapidly evolving viruses such as influenza and SARS-CoV-2. Precise measurement of the extent to which imprinting/OAS inhibits the recruitment of new B cell clones by boosting is challenging because cellular and temporal origins cannot readily be assigned to antibodies in circulation. Thus, the extent to which imprinting/OAS impacts the induction of new responses in various settings remains unclear. To address this, we developed a "molecular fate-mapping" approach in which serum antibodies derived from specific cohorts of B cells can be differentially detected. We show that, upon sequential homologous boosting, the serum antibody response strongly favors reuse of the first cohort of B cell clones over the recruitment of new, naive-derived B cells. This "primary addiction" decreases as a function of antigenic distance, allowing secondary immunization with divergent influenza virus or SARS-CoV-2 glycoproteins to overcome imprinting/OAS by targeting novel epitopes absent from the priming variant. Our findings have implications for the understanding of imprinting/OAS, and for the design and testing of vaccines aimed at eliciting antibodies to evolving antigens.

Antigen presentation dynamics shape the response to emergent variants like SARS-CoV-2 Omicron strain after multiple vaccinations with wild type strain (preprint)

The Omicron variant of SARS-CoV-2 evades neutralization by most serum antibodies elicited by two doses of mRNA vaccines, but a third dose of the same vaccine increases anti-Omicron neutralizing antibodies. By combining computational modeling with data from vaccinated humans we reveal mechanisms underlying this observation. After the first dose, limited antigen availability in germinal centers results in a response dominated by B cells with high germline affinities for immunodominant epitopes that are significantly mutated in an Omicron-like variant. After the second dose, expansion of these memory cells and differentiation into plasma cells shape antibody responses that are thus ineffective for such variants. However, in secondary germinal centers, pre-existing higher affinity antibodies mediate enhanced antigen presentation and they can also partially mask dominant epitopes. These effects generate memory B cells that target subdominant epitopes that are less mutated in Omicron. The third dose expands these cells and boosts anti-variant neutralizing antibodies.

Human anti-ACE2 monoclonal antibodies as pan-sarbecovirus prophylactic agents (preprint)

Human monoclonal antibodies from convalescent individuals that target the SARS-CoV-2 spike protein have been deployed as therapeutics against SARS-CoV-2. However, nearly all of these antibodies have been rendered obsolete by SARS-CoV-2 variants that evolved to resist similar, naturally occurring antibodies. Here, we describe the development of human monoclonal antibodies that bind the ACE2 receptor rather than the viral spike protein. These antibodies block infection by all ACE2 binding sarbecoviruses, including emergent SARS-CoV-2 variants. Structural and biochemical analyses revealed that the antibodies target an ACE2 epitope that engages SARS-CoV-2 spike. Importantly, the antibodies do not inhibit ACE2 enzymatic activity, nor do they induce ACE depletion from cell surfaces. The antibodies exhibit favorable pharmacology and protect human ACE2 knock-in mice against SARS-CoV-2 infection. Such antibodies should be useful prophylactic and treatment agents against any current and future SARS-CoV-2 variants, as well as ACE2-binding sarbecoviruses that might emerge as future pandemic threats

Epistasis lowers the genetic barrier to SARS-CoV-2 neutralizing antibody escape (preprint)

Consecutive waves of SARS-CoV-2 infection have been driven in part by the repeated emergence of variants with mutations that confer resistance to neutralizing antibodies Nevertheless, prolonged or repeated antigen exposure generates diverse memory B-cells that can produce affinity matured receptor binding domain (RBD)-specific antibodies that likely contribute to ongoing protection against severe disease. To determine how SARS-CoV-2 omicron variants might escape these broadly neutralizing antibodies, we subjected chimeric viruses encoding spike proteins from ancestral, BA.1 or BA.2 variants to selection pressure by a collection of 40 broadly neutralizing antibodies from individuals with various SARS-CoV-2 antigen exposures. Notably, pre-existing substitutions in the BA.1 and BA.2 spikes facilitated acquisition of resistance to many broadly neutralizing antibodies. Specifically, selection experiments identified numerous RBD substitutions that did not confer resistance to broadly neutralizing antibodies in the context of the ancestral Wuhan-Hu-1 spike sequence, but did so in the context of BA.1 and BA.2. A subset of these substitutions corresponds to those that have appeared in several BA.2 daughter lineages that have recently emerged, such as BA.5. By including as few as 2 or 3 of these additional changes in the context of BA.5, we generated spike proteins that were resistant to nearly all of the 40 broadly neutralizing antibodies and were poorly neutralized by plasma from most individuals. The emergence of omicron variants has therefore not only allowed SARS-CoV-2 escape from previously elicited neutralizing antibodies but also lowered the genetic barrier to the acquisition of resistance to the subset of antibodies that remained effective against early omicron variants.

Memory B cell responses to Omicron subvariants after SARS-CoV-2 mRNA breakthrough infection (preprint)

Individuals that receive a 3rd mRNA vaccine dose show enhanced protection against severe COVID19 but little is known about the impact of breakthrough infections on memory responses. Here, we examine the memory antibodies that develop after a 3rd or 4th antigenic exposure by Delta or Omicron BA.1 infection, respectively. A 3rd exposure to antigen by Delta breakthrough increases the number of memory B cells that produce antibodies with comparable potency and breadth to a 3rd mRNA vaccine dose. A 4th antigenic exposure with Omicron BA.1 infection increased variant specific plasma antibody and memory B cell responses. However, the 4th exposure did not increase the overall frequency of memory B cells or their general potency or breadth compared to a 3rd mRNA vaccine dose. In conclusion, a 3rd antigenic exposure by Delta infection elicits strain-specific memory responses and increases in the overall potency and breadth of the memory B cells. In contrast, the effects of a 4th antigenic exposure with Omicron BA.1 is limited to increased strain specific memory with little effect on the potency or breadth of memory B cell antibodies. The results suggest that the effect of strain-specific boosting on memory B cell compartment may be limited.

Antibody feedback regulation of memory B cell development in SARS-CoV-2 mRNA vaccination (preprint)

Feedback inhibition of humoral immunity by antibodies was initially documented in guinea pigs by Theobald Smith in 1909, who showed that passive administration of excess anti-Diphtheria toxin inhibited immune responses1. Subsequent work documented that antibodies can enhance or inhibit immune responses depending on antibody isotype, affinity, the physical nature of the antigen, and engagement of immunoglobulin (Fc) and complement (C) receptors2, 3. However, little is known about how pre-existing antibodies might influence the subsequent development of memory B cells. Here we examined the memory B cell response in individuals who received two high-affinity IgG1 anti-SARS-CoV-2 receptor binding domain (RBD)-specific monoclonal antibodies, C144-LS and C135-LS, and subsequently two doses of a SARS-CoV-2 mRNA vaccine. The two antibodies target Class 2 and 3 epitopes that dominate the initial immune response to SARS-CoV-2 infection and mRNA vaccination4-8. Antibody responses to the vaccine in C144-LS and C135-LS recipients produced plasma antigen binding and neutralizing titers that were fractionally lower but not statistically different to controls. In contrast, memory B cells enumerated by flow cytometry after the second vaccine dose were present in higher numbers than in controls. However, the memory B cells that developed in antibody recipients differed from controls in that they were not enriched in VH3-53, VH1-46 and VH3-66 genes and predominantly expressed low-affinity IgM antibodies that carried small numbers of somatic mutations. These antibodies showed altered RBD target specificity consistent with epitope masking, and only 1 out of 77 anti-RBD memory antibodies tested neutralized the virus. The results indicate that pre-existing high-affinity antibodies bias memory B cell selection and have a profound effect on the development of immunological memory in humans that may in part explain the shifting target profile of memory antibodies elicited by the 3rd mRNA vaccine dose.

Inhibition of major histocompatibility complex-I antigen presentation by sarbecovirus ORF7a proteins (preprint)

Viruses employ a variety of strategies to escape or counteract immune responses, including depletion of cell surface major histocompatibility complex class I (MHC-I), that would ordinarily present viral peptides to CD8+ cytotoxic T cells. As part of a screen to elucidate biological activities associated with individual SARS-CoV-2 viral proteins, we found that ORF7a reduced cell surface MHC-I levels by approximately 5-fold. Nevertheless, in cells infected with SARS-CoV-2, surface MHC-I levels were reduced even in the absence of ORF7a, suggesting additional mechanisms of MHC-I downregulation. ORF7a proteins from a sample of sarbecoviruses varied in their ability to induce MHC-I downregulation and, unlike SARS-CoV-2, the ORF7a protein from SARS-CoV lacked MHC-I downregulating activity. A single-amino acid at position 59 (T/F) that is variable among sarbecovirus ORF7a proteins governed the difference in MHC-I downregulating activity. SARS-CoV-2 ORF7a physically associated with the MHC-I heavy chain and inhibited the presentation of expressed antigen to CD8+ T-cells. Speficially, ORF7a prevented the assembly of the MHC-I peptide loading complex and causing retention of MHC-I in the endoplasmic reticulum. The differential ability of ORF7a proteins to function in this way might affect sarbecovirus dissemination and persistence in human populations, particularly those with infection- or vaccine-elicited immunity.

Humoral immunity to SARS-CoV-2 elicited by combination COVID-19 vaccination regimens (preprint)

The SARS-CoV-2 pandemic prompted a global vaccination effort and the development of numerous COVID-19 vaccines at an unprecedented scale and pace. As a result, current COVID-19 vaccination regimens comprise diverse vaccine modalities, immunogen combinations and dosing intervals. Here, we compare vaccine-specific antibody and memory B cell responses following two-dose mRNA, single-dose Ad26.COV2.S and two-dose ChAdOx1 or combination ChAdOx1/mRNA vaccination. Plasma neutralizing activity as well as the magnitude, clonal composition and antibody maturation of the RBD-specific memory B cell compartment showed substantial differences between the vaccination regimens. While individual monoclonal antibodies derived from memory B cells exhibited similar binding affinities and neutralizing potency against Wuhan-Hu-1 SARS-CoV-2, there were significant differences in epitope specificity and neutralizing breadth against viral variants of concern. Although the ChAdOx1 vaccine was inferior to mRNA and Ad26.COV2.S in several respects, biochemical and structural analyses revealed enrichment in a subgroup of memory B cell neutralizing antibodies with distinct RBD-binding properties resulting in remarkable potency and breadth.

Antibody evolution to SARS-CoV-2 after single-dose Ad26.COV2.S vaccine (preprint)

The single dose Ad.26.COV.2 (Janssen) vaccine elicits lower levels of neutralizing antibodies and shows more limited efficacy in protection against infection than either of the available mRNA vaccines. In addition, the Ad.26.COV.2 has been less effective in protection against severe disease during the Omicron surge. Here, we examined the memory B cell response to single dose Ad.26.COV.2 vaccination. Compared to mRNA vaccines, Ad.26.COV.2 recipients had significantly lower numbers of RBD-specific memory B cells 1.5 or 6 months after vaccination. Memory antibodies elicited by both vaccine types show comparable neutralizing potency against SARS-CoV-2 and Delta. However, the number of memory cells producing Omicron neutralizing antibodies was somewhat lower after Ad.26.COV.2 than mRNA vaccination. The data help explain why boosting Ad.26.COV.2 vaccine recipients with mRNA vaccines is effective, and why the Janssen vaccine appears to have been less protective against severe disease during the Omicron surge than the mRNA vaccine.

Increased Potency and Breadth of SARS-CoV-2 Neutralizing Antibodies After a Third mRNA Vaccine Dose (preprint)

The omicron variant of SARS-CoV-2 infected very large numbers of SARS-CoV-2 vaccinated and convalescent individuals. The penetrance of this variant in the antigen experienced human population can be explained in part by the relatively low levels of plasma neutralizing activity against Omicron in people who were infected or vaccinated with the original Wuhan-Hu-1 strain. The 3rd mRNA vaccine dose produces an initial increase in circulating anti-Omicron neutralizing antibodies, but titers remain 10-20-fold lower than against Wuhan-Hu-1 and are, in many cases, insufficient to prevent infection. Despite the reduced protection from infection, individuals that received 3 doses of an mRNA vaccine were highly protected from the more serious consequences of infection. Here we examine the memory B cell repertoire in a longitudinal cohort of individuals receiving 3 mRNA vaccine doses. We find that the 3rd dose is accompanied by an increase in, and evolution of, anti-receptor binding domain specific memory B cells. The increase is due to expansion of memory B cell clones that were present after the 2nd vaccine dose as well as the emergence of new clones. The antibodies encoded by these cells showed significantly increased potency and breadth when compared to antibodies obtained after the 2nd vaccine dose. Notably, the increase in potency was especially evident among newly developing clones of memory cells that differed from the persisting clones in targeting more conserved regions of the RBD. Overall, more than 50% of the analyzed neutralizing antibodies in the memory compartment obtained from individuals receiving a 3rd mRNA vaccine dose neutralized Omicron. Thus, individuals receiving 3 doses of an mRNA vaccine encoding Wuhan-Hu-1, have a diverse memory B cell repertoire that can respond rapidly and produce antibodies capable of clearing even diversified variants such as Omicron. These data help explain why a 3rd dose of an mRNA vaccine that was not specifically designed to protect against variants is effective against variant-induced serious disease.

VPS29 exerts opposing effects on endocytic viral entry (preprint)

Emerging zoonotic viral pathogens threaten global health and there is an urgent need to discover host and viral determinants influencing infection. We performed a loss-of-function genome-wide CRISPR screen in a human lung cell line using HCoV-OC43, a human betacoronavirus. One candidate gene, VPS29, was required for infection by HCoV-OC43, SARS-CoV-2, other endemic and pandemic threat coronaviruses as well as ebolavirus. However, VPS29 deficiency had no effect on certain other viruses that enter cells via endosomes and had an opposing, enhancing effect on influenza A virus infection. VPS29 deficiency caused changes endosome morphology, and acidity and attenuated the activity of endosomal proteases. These changes in endosome properties caused incoming coronavirus, but not influenza virus particles, to become entrapped therein. Overall, these data show how host regulation of endosome characteristics can influence viral susceptibility and identify a host pathway that could serve as a pharmaceutical target for intervention in zoonotic viral diseases.

Antibody Evolution after SARS-CoV-2 mRNA Vaccination (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection produces B-cell responses that continue to evolve for at least one year. During that time, memory B cells express increasingly broad and potent antibodies that are resistant to mutations found in variants of concern. As a result, vaccination of coronavirus disease 2019 (COVID-19) convalescent individuals with currently available mRNA vaccines produces high levels of plasma neutralizing activity against all variants tested. Here, we examine memory B cell evolution 5 months after vaccination with either Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) mRNA vaccines in a cohort of SARS-CoV-2 naive individuals. Between prime and boost, memory B cells produce antibodies that evolve increased neutralizing activity, but there is no further increase in potency or breadth thereafter. Instead, memory B cells that emerge 5 months after vaccination of naive individuals express antibodies that are equivalent to those that dominate the initial response. We conclude that memory antibodies selected over time by natural infection have greater potency and breadth than antibodies elicited by vaccination. These results suggest that boosting vaccinated individuals with currently available mRNA vaccines would produce a quantitative increase in plasma neutralizing activity but not the qualitative advantage against variants obtained by vaccinating convalescent individuals.

Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies (preprint)

Many anti-SARS-CoV-2 neutralizing antibodies target the ACE2-binding site on viral spike receptor-binding domains (RBDs). The most potent antibodies recognize exposed variable epitopes, often rendering them ineffective against other sarbecoviruses and SARS-CoV-2 variants. Class 4 anti-RBD antibodies against a less-exposed, but more-conserved, cryptic epitope could recognize newly-emergent zoonotic sarbecoviruses and variants, but usually show only weak neutralization potencies. We characterized two class 4 anti-RBD antibodies derived from COVID-19 donors that exhibited broad recognition and potent neutralization of zoonotic coronavirus and SARS-CoV-2 variants. C118-RBD and C022-RBD structures revealed CDRH3 mainchain H-bond interactions that extended an RBD {beta}-sheet, thus reducing sensitivity to RBD sidechain changes, and epitopes that extended from the cryptic epitope to occlude ACE2 binding. A C118-spike trimer structure revealed rotated RBDs to allow cryptic epitope access and the potential for intra-spike crosslinking to increase avidity. These studies facilitate vaccine design and illustrate potential advantages of class 4 RBD-binding antibody therapeutics.

Nanobody Repertoires for Exposing Vulnerabilities of SARS-CoV-2 (preprint)

Despite the great promise of vaccines, the COVID-19 pandemic is ongoing and future serious outbreaks are highly likely, so that multi-pronged containment strategies will be required for many years. Nanobodies are the smallest naturally occurring single domain antigen binding proteins identified to date, possessing numerous properties advantageous to their production and use. We present a large repertoire of high affinity nanobodies against SARS-CoV-2 Spike protein with excellent kinetic and viral neutralization properties, which can be strongly enhanced with oligomerization. This repertoire samples the epitope landscape of the Spike ectodomain inside and outside the receptor binding domain, recognizing a multitude of distinct epitopes and revealing multiple neutralization targets of pseudoviruses and authentic SARS-CoV-2, including in primary human airway epithelial cells. Combinatorial nanobody mixtures show highly synergistic activities, and are resistant to mutational escape and emerging viral variants of concern. These nanobodies establish an exceptional resource for superior COVID-19 prophylactics and therapeutics.

Mutational escape from the polyclonal antibody response to SARS-CoV-2 infection is largely shaped by a single class of antibodies (preprint)

Monoclonal antibodies targeting a variety of epitopes have been isolated from individuals previously infected with SARS-CoV-2, but the relative contributions of these different antibody classes to the polyclonal response remains unclear. Here we use a yeast-display system to map all mutations to the viral spike receptor-binding domain (RBD) that escape binding by representatives of three potently neutralizing classes of anti-RBD antibodies with high-resolution structures. We compare the antibody-escape maps to similar maps for convalescent polyclonal plasma, including plasma from individuals from whom some of the antibodies were isolated. The plasma-escape maps most closely resemble those of a single class of antibodies that target an epitope on the RBD that includes site E484. Therefore, although the human immune system can produce antibodies that target diverse RBD epitopes, in practice the polyclonal response to infection is dominated by a single class of antibodies targeting an epitope that is already undergoing rapid evolution.

Multimeric nanobodies from camelid engineered mice and llamas potently neutralize SARS-CoV-2 variants (preprint)

Since the start of the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused more than 2 million deaths worldwide. Many vaccines have been deployed to date, but the continual evolution of the viral receptor-binding domain (RBD) has recently challenged their efficacy. In particular, SARS-CoV-2 variants originating in the U.K. (B.1.1.7), South Africa (B.1.351) and New York (B.1.526) have reduced neutralization activity from convalescent sera and compromised the efficacy of some antibody cocktails that received emergency use authorization. Whereas vaccines can be updated periodically to account for emerging variants, complementary strategies are urgently needed to avert viral escape. One potential alternative is the use of camelid VHHs (also known as nanobodies), which due to their small size can recognize protein domains that are often inaccessible to conventional antibodies. Here, we isolate anti-RBD nanobodies from llamas and nanomice we engineered to produce VHHs cloned from alpacas, dromedaries and camels. Through binding assays and cryo-electron microscopy, we identified two sets of highly neutralizing nanobodies. The first group expresses VHHs that circumvent RBD antigenic drift by recognizing a region outside the ACE2-binding site that is conserved in coronaviruses but is not typically targeted by monoclonal antibodies. The second group is almost exclusively focused to the RBD-ACE2 interface and fails to neutralize pseudoviruses carrying the E484K or N501Y substitutions. Notably however, they do neutralize the RBD variants when expressed as homotrimers, rivaling the most potent antibodies produced to date against SARS-CoV-2. These findings demonstrate that multivalent nanobodies overcome SARS-CoV-2 variant mutations through two separate mechanisms: enhanced avidity for the ACE2 binding domain, and recognition of conserved epitopes largely inaccessible to human antibodies. Therefore, while new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.

Rapid protection from COVID-19 in nonhuman primates vaccinated intramuscularly but not intranasally with a single dose of a recombinant vaccine (preprint)

The ongoing pandemic of Coronavirus disease 2019 (COVID-19) continues to exert a significant burden on health care systems worldwide. With limited treatments available, vaccination remains an effective strategy to counter transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recent discussions concerning vaccination strategies have focused on identifying vaccine platforms, number of doses, route of administration, and time to reach peak immunity against SARS-CoV-2. Here, we generated a single dose, fast-acting vesicular stomatitis virus-based vaccine derived from the licensed Ebola virus (EBOV) vaccine rVSV-ZEBOV, expressing the SARS-CoV-2 spike protein and the EBOV glycoprotein (VSV-SARS2-EBOV). Rhesus macaques vaccinated intramuscularly (IM) with a single dose of VSV-SARS2-EBOV were protected within 10 days and did not show signs of COVID-19 pneumonia. In contrast, IN vaccination resulted in limited immunogenicity and enhanced COVID-19 pneumonia compared to control animals. While IM and IN vaccination both induced neutralizing antibody titers, only IM vaccination resulted in a significant cellular immune response. RNA sequencing data bolstered these results by revealing robust activation of the innate and adaptive immune transcriptional signatures in the lungs of IM-vaccinated animals only. Overall, the data demonstrates that VSV-SARS2-EBOV is a potent single-dose COVID-19 vaccine candidate that offers rapid protection based on the protective efficacy observed in our study.

Interferon-regulated genetic programs and JAK/STAT pathway activate the intronic promoter of the short ACE2 isoform in renal proximal tubules (preprint)

Summary: Recently, a short, interferon-inducible isoform of Angiotensin-Converting Enzyme 2 (ACE2), dACE2 was identified. ACE2 is a SARS-Cov-2 receptor and changes in its renal expression have been linked to several human nephropathies. These changes were never analyzed in context of dACE2, as its expression was not investigated in the kidney. We used Human Primary Proximal Tubule (HPPT) cells to show genome-wide gene expression patterns after cytokine stimulation, with emphasis on the ACE2/dACE2 locus. Putative regulatory elements controlling dACE2 expression were identified using ChIP-seq and RNA-seq. qRT-PCR differentiating between ACE2 and dACE2 revealed 300- and 600-fold upregulation of dACE2 by IFN and IFN{beta}, respectively, while full length ACE2 expression was almost unchanged. JAK inhibitor ruxolitinib ablated STAT1 and dACE2 expression after interferon treatment. Finally, with RNA-seq, we identified a set of genes, largely immune-related, induced by cytokine treatment. These gene expression profiles provide new insights into cytokine response of proximal tubule cells.

An all-solid-state heterojunction oxide transistor for the rapid detection of biomolecules and SARS-CoV-2 spike S1 protein (preprint)

Solid-state transistor sensors that can detect biomolecules in real time are highly attractive for emerging bioanalytical applications. However, combining cost-effective manufacturing with high sensitivity, specificity and fast sensing response, remains challenging. Here we develop low-temperature solution-processed In2O3/ZnO heterojunction transistors featuring a geometrically engineered tri-channel architecture for rapid real-time detection of different biomolecules. The sensor combines a high electron mobility channel, attributed to the quasi-two-dimensional electron gas (q2DEG) at the buried In2O3/ZnO heterointerface, in close proximity to a sensing surface featuring tethered analyte receptors. The unusual tri-channel design enables strong coupling between the buried q2DEG and the minute electronic perturbations occurring during receptor-analyte interactions allowing for robust, real-time detection of biomolecules down to attomolar (aM) concentrations. By functionalizing the tri-channel surface with SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) antibody receptors, we demonstrate real-time detection of the SARS-CoV-2 spike S1 protein down to attomolar concentrations in under two minutes.