Vaccination induces broadly neutralizing antibody precursors to HIV gp41.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is continuing to disrupt personal lives, global healthcare systems, and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission, and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits, and cynomolgus macaques. The vaccine-induced immunity protects macaques against a high-dose challenge, resulting in strongly reduced viral infection and replication in the upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.

Human immunoglobulin repertoire analysis guides design of vaccine priming immunogens targeting HIV V2-apex broadly neutralizing antibody precursors.

Broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env) V2-apex region are important leads for HIV vaccine design. Most V2-apex bnAbs engage Env with an uncommonly long heavy-chain complementarity-determining region 3 (HCDR3), suggesting that the rarity of bnAb precursors poses a challenge for vaccine priming. We created precursor sequence definitions for V2-apex HCDR3-dependent bnAbs and searched for related precursors in human antibody heavy-chain ultradeep sequencing data from 14 HIV-unexposed donors. We found potential precursors in a majority of donors for only two long-HCDR3 V2-apex bnAbs, PCT64 and PG9, identifying these bnAbs as priority vaccine targets. We then engineered ApexGT Env trimers that bound inferred germlines for PCT64 and PG9 and had higher affinities for bnAbs, determined cryo-EM structures of ApexGT trimers complexed with inferred-germline and bnAb forms of PCT64 and PG9, and developed an mRNA-encoded cell-surface ApexGT trimer. These methods and immunogens have promise to assist HIV vaccine development.

Glycan heterogeneity as a cause of the persistent fraction in HIV-1 neutralization.

Neutralizing antibodies (NAbs) to multiple epitopes on the HIV-1-envelope glycoprotein (Env) have been isolated from infected persons. The potency of NAbs is measured more often than the size of the persistent fraction of infectivity at maximum neutralization, which may also influence preventive efficacy of active or passive immunization and the therapeutic outcome of the latter. Many NAbs neutralize HIV-1 CZA97.012, a clone of a Clade-C isolate, to ~100%. But here NAb PGT151, directed to a fusion-peptide epitope, left a persistent fraction of 15%. NAb PGT145, ligating the Env-trimer apex, left no detectable persistent fraction. The divergence in persistent fractions was further analyzed by depletion of pseudoviral populations of the most PGT151- and PGT145-reactive virions. Thereby, neutralization by the non-depleting NAb increased, whereas neutralization by the depleting NAb decreased. Furthermore, depletion by PGT151 increased sensitivity to autologous neutralization by sera from rabbits immunized with soluble native-like CZA97.012 trimer: substantial persistent fractions were reduced. NAbs in these sera target epitopes comprising residue D411 at the V4-ß19 transition in a defect of the glycan shield on CZA97.012 Env. NAb binding to affinity-fractionated soluble native-like CZA97.012 trimer differed commensurately with neutralization in analyses by ELISA and surface plasmon resonance. Glycan differences between PGT151- and PGT145-purified trimer fractions were then demonstrated by mass spectrometry, providing one explanation for the differential antigenicity. These differences were interpreted in relation to a new structure at 3.4-Å resolution of the soluble CZA97.012 trimer determined by cryo-electron microscopy. The trimer adopted a closed conformation, refuting apex opening as the cause of reduced PGT145 binding to the PGT151-purified form. The evidence suggests that differences in binding and neutralization after trimer purification or pseudovirus depletion with PGT145 or PGT151 are caused by variation in glycosylation, and that some glycan variants affect antigenicity through direct effects on antibody contacts, whereas others act allosterically.

The Glycan Hole Area of HIV-1 Envelope Trimers Contributes Prominently to the Induction of Autologous Neutralization.

The human immunodeficiency virus type 1 (HIV-1) trimeric envelope glycoprotein (Env) is heavily glycosylated, creating a dense glycan shield that protects the underlying peptidic surface from antibody recognition. The absence of conserved glycans, due to missing potential N-linked glycosylation sites (PNGS), can result in strain-specific, autologous neutralizing antibody (NAb) responses. Here, we sought to gain a deeper understanding of the autologous neutralization by introducing holes in the otherwise dense glycan shields of the AMC011 and AMC016 SOSIP trimers. Specifically, when we knocked out the N130 and N289 glycans, which are absent from the well-characterized B41 SOSIP trimer, we observed stronger autologous NAb responses. We also analyzed the highly variable NAb responses induced in rabbits by diverse SOSIP trimers from subtypes A, B, and C. Statistical analysis, using linear regression, revealed that the cumulative area exposed on a trimer by glycan holes correlates with the magnitude of the autologous NAb response. IMPORTANCE Forty years after the first description of HIV-1, the search for a protective vaccine is still ongoing. The sole target for antibodies that can neutralize the virus are the trimeric envelope glycoproteins (Envs) located on the viral surface. The glycoprotein surface is covered with glycans that shield off the underlying protein components from recognition by the immune system. However, the Env trimers of some viral strains have holes in the glycan shield. Immunized animals developed antibodies against such glycan holes. These antibodies are generally strain specific. Here, we sought to gain a deeper understanding of what drives these specific immune responses. First, we show that strain-specific neutralizing antibody responses can be increased by creating artificial holes in the glycan shield. Second, when studying a diverse set of Env trimers with different characteristics, we found that the surface area of the glycan holes contributes prominently to the induction of strain-specific neutralizing antibodies.

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens.

HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets-the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus (PV) assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and eliminate other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-NAbs, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the C-strand. Notably, a D167N mutation improved V2-sensitivity in several cases. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing glycans at other positions frequently had global "ripple" effects on glycan maturation and sequon occupation throughout the gp120 outer domain and gp41. V2 MAb CH01 selectively bound to trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a rare N49 glycan was found to perturb gp41 glycans, increasing FP NAb sensitivity-and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30-40 spikes. Taken together, we identified 7 diverse trimers with a range of sensitivities to two targets to allow rigorous testing of immunofocusing vaccine concepts.

A cross-neutralizing antibody between HIV-1 and influenza virus.

Incessant antigenic evolution enables the persistence and spread of influenza virus in the human population. As the principal target of the immune response, the hemagglutinin (HA) surface antigen on influenza viruses continuously acquires and replaces N-linked glycosylation sites to shield immunogenic protein epitopes using host-derived glycans. Anti-glycan antibodies, such as 2G12, target the HIV-1 envelope protein (Env), which is even more extensively glycosylated and contains under-processed oligomannose-type clusters on its dense glycan shield. Here, we illustrate that 2G12 can also neutralize human seasonal influenza A H3N2 viruses that have evolved to present similar oligomannose-type clusters on their HAs from around 20 years after the 1968 pandemic. Using structural biology and mass spectrometric approaches, we find that two N-glycosylation sites close to the receptor binding site (RBS) on influenza hemagglutinin represent the oligomannose cluster recognized by 2G12. One of these glycan sites is highly conserved in all human H3N2 strains and the other emerged during virus evolution. These two N-glycosylation sites have also become crucial for fitness of recent H3N2 strains. These findings shed light on the evolution of the glycan shield on influenza virus and suggest 2G12-like antibodies can potentially act as broad neutralizers to target human enveloped viruses.

Insertion of atypical glycans into the tumor antigen-binding site identifies DLBCLs with distinct origin and behavior.

Glycosylation of the surface immunoglobulin (Ig) variable region is a remarkable follicular lymphoma-associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCLs) that acquire N-glycosylation sites selectively in the Ig complementarity-determining regions (CDRs) of the antigen-binding sites. Mass spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures because they are buried in the CDR loops. Acquisition of sites occurs in ∼50% of germinal-center B-cell-like DLBCL (GCB-DLBCL), mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell-like DLBCL Ig, which rarely has sites in the CDR and does not seem to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites are associated with more rapid disease progression and with significant gene set enrichment of the B-cell receptor, PI3K/AKT/MTORC1 pathway, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate recognition domain. Oligomannosylation of the tumor Ig is a posttranslational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and it could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN-expressing M2-polarized macrophages.

Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens.

Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Computational design of protein nanoparticles with differing sizes and geometries enables combination with antigens of choice to test novel multimerization concepts in immunization strategies where the goal is to improve the induction and maturation of neutralizing antibody lineages. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to an underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. When the immunogenicity of ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle or as soluble proteins were compared in rabbits, the two immunogens elicited similar serum antibody binding titers against the trimer component. Neutralizing antibody titers were slightly elevated in the animals given the nanoparticle immunogen and were initially more focused to the trimer apex. Altogether, our findings indicate that tetrahedral nanoparticles can be successfully applied for presentation of HIV Env trimer immunogens; however, the optimal implementation to different immunization strategies remains to be determined.

Augmenting glycosylation-directed folding pathways enhances the fidelity of HIV Env immunogen production in plants.

Heterologous glycoprotein production relies on host glycosylation-dependent folding. When the biosynthetic machinery differs from the usual expression host, there is scope to remodel the assembly pathway to enhance glycoprotein production. Here we explore the integration of chaperone coexpression with glyco-engineering to improve the production of a model HIV-1 envelope antigen. Calreticulin was coexpressed to support protein folding together with Leishmania major STT3D oligosaccharyltransferase, to improve glycan occupancy, RNA interference to suppress the formation of truncated glycans, and Nicotiana benthamiana plants lacking α1,3-fucosyltransferase and ß1,2-xylosyltransferase was used as an expression host to prevent plant-specific complex N-glycans forming. This approach reduced the formation of undesired aggregates, which predominated in the absence of glyco-engineering. The resulting antigen also exhibited increased glycan occupancy, albeit to a slightly lower level than the equivalent mammalian cell-produced protein. The antigen was decorated almost exclusively with oligomannose glycans, which were less processed compared with the mammalian protein. Immunized rabbits developed comparable immune responses to the plant-produced and mammalian cell-derived antigens, including the induction of autologous neutralizing antibodies when the proteins were used to boost DNA and modified vaccinia Ankara virus-vectored vaccines. This study demonstrates that engineering glycosylation-directed folding offers a promising route to enhance the production of complex viral glycoproteins in plants.

Site-Specific Steric Control of SARS-CoV-2 Spike Glycosylation.

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity among the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against S protein from infectious virus, cultured in Vero cells. We find patterns that are conserved across all samples, and this can be associated with site-specific stalling of glycan maturation that acts as a highly sensitive reporter of protein structure. Molecular dynamics simulations of a fully glycosylated spike support a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.

Development of a high-sensitivity ELISA detecting IgG, IgA and IgM antibodies to the SARS-CoV-2 spike glycoprotein in serum and saliva.

Detecting antibody responses during and after SARS-CoV-2 infection is essential in determining the seroepidemiology of the virus and the potential role of antibody in disease. Scalable, sensitive and specific serological assays are essential to this process. The detection of antibody in hospitalized patients with severe disease has proven relatively straightforward; detecting responses in subjects with mild disease and asymptomatic infections has proven less reliable. We hypothesized that the suboptimal sensitivity of antibody assays and the compartmentalization of the antibody response may contribute to this effect. We systematically developed an ELISA, optimizing different antigens and amplification steps, in serum and saliva from non-hospitalized SARS-CoV-2-infected subjects. Using trimeric spike glycoprotein, rather than nucleocapsid, enabled detection of responses in individuals with low antibody responses. IgG1 and IgG3 predominate to both antigens, but more anti-spike IgG1 than IgG3 was detectable. All antigens were effective for detecting responses in hospitalized patients. Anti-spike IgG, IgA and IgM antibody responses were readily detectable in saliva from a minority of RT-PCR confirmed, non-hospitalized symptomatic individuals, and these were mostly subjects who had the highest levels of anti-spike serum antibodies. Therefore, detecting antibody responses in both saliva and serum can contribute to determining virus exposure and understanding immune responses after SARS-CoV-2 infection.

Suppression of O-Linked Glycosylation of the SARS-CoV-2 Spike by Quaternary Structural Restraints.

Understanding the glycosylation of the envelope spike (S) protein of SARS-CoV-2 is important in defining the antigenic surface of this key viral target. However, the underlying protein architecture may significantly influence glycan occupancy and processing. There is, therefore, potential for different recombinant fragments of S protein to display divergent glycosylation. Here, we show that the receptor binding domain (RBD), when expressed as a monomer, exhibits O-linked glycosylation, which is not recapitulated in the native-like soluble trimeric protein. We unambiguously assign O-linked glycosylation by homogenizing N-linked glycosylation using the enzymatic inhibitor, kifunensine, and then analyzing the resulting structures by electron-transfer higher-energy collision dissociation (EThcD) in an Orbitrap Eclipse Tribrid instrument. In the native-like trimer, we observe a single unambiguous O-linked glycan at T323, which displays very low occupancy. In contrast, several sites of O-linked glycosylation can be identified when RBD is expressed as a monomer, with T323 being almost completely occupied. We ascribe this effect to the relaxation of steric restraints arising from quaternary protein architecture. Our analytical approach has also highlighted that fragmentation ions arising from trace levels of truncated N-linked glycans can be misassigned as proximal putative O-linked glycan structures, particularly where a paucity of diagnostic fragments were obtained. Overall, we show that in matched expression systems the quaternary protein architecture limits O-linked glycosylation of the spike protein.

Neutralizing Antibody Responses Induced by HIV-1 Envelope Glycoprotein SOSIP Trimers Derived from Elite Neutralizers.

The induction of broadly neutralizing antibodies (bNAbs) is a major goal in vaccine research. HIV-1-infected individuals that develop exceptionally strong bNAb responses, termed elite neutralizers, can inform vaccine design by providing blueprints for the induction of similar bNAb responses. We describe a new recombinant native-like envelope glycoprotein (Env) SOSIP trimer, termed AMC009, based on the viral founder sequences of an elite neutralizer. The subtype B AMC009 SOSIP protein formed stable native-like trimers that displayed multiple bNAb epitopes. Overall, its structure at 4.3-Å resolution was similar to that of BG505 SOSIP.664. The AMC009 trimer resembled one from a second elite neutralizer, AMC011, in having a dense and complete glycan shield. When tested as immunogens in rabbits, the AMC009 trimers did not induce autologous neutralizing antibody (NAb) responses efficiently while the AMC011 trimers did so very weakly, outcomes that may reflect the completeness of their glycan shields. The AMC011 trimer induced antibodies that occasionally cross-neutralized heterologous tier 2 viruses, sometimes at high titer. Cross-neutralizing antibodies were more frequently elicited by a trivalent combination of AMC008, AMC009, and AMC011 trimers, all derived from subtype B viruses. Each of these three individual trimers could deplete the NAb activity from the rabbit sera. Mapping the polyclonal sera by electron microscopy revealed that antibodies of multiple specificities could bind to sites on both autologous and heterologous trimers. These results advance our understanding of how to use Env trimers in multivalent vaccination regimens and the immunogenicity of trimers derived from elite neutralizers.IMPORTANCE Elite neutralizers, i.e., individuals who developed unusually broad and potent neutralizing antibody responses, might serve as blueprints for HIV-1 vaccine design. Here, we studied the immunogenicity of native-like recombinant envelope glycoprotein (Env) trimers based on viral sequences from elite neutralizers. While immunization with single trimers from elite neutralization did not recapitulate the breadth and potency of neutralization observed in these infected individuals, a combination of three subtype B Env trimers from elite neutralizers resulted in some neutralization breadth within subtype B viruses. These results should guide future efforts to design vaccines to induce broadly neutralizing antibodies.

Similarities and differences between native HIV-1 envelope glycoprotein trimers and stabilized soluble trimer mimetics.

The HIV-1 envelope glycoprotein (Env) trimer is located on the surface of the virus and is the target of broadly neutralizing antibodies (bNAbs). Recombinant native-like soluble Env trimer mimetics, such as SOSIP trimers, have taken a central role in HIV-1 vaccine research aimed at inducing bNAbs. We therefore performed a direct and thorough comparison of a full-length unmodified Env trimer containing the transmembrane domain and the cytoplasmic tail, with the sequence matched soluble SOSIP trimer, both based on an early Env sequence (AMC011) from an HIV+ individual that developed bNAbs. The structures of the full-length AMC011 trimer bound to either bNAb PGT145 or PGT151 were very similar to the structures of SOSIP trimers. Antigenically, the full-length and SOSIP trimers were comparable, but in contrast to the full-length trimer, the SOSIP trimer did not bind at all to non-neutralizing antibodies, most likely as a consequence of the intrinsic stabilization of the SOSIP trimer. Furthermore, the glycan composition of full-length and SOSIP trimers was similar overall, but the SOSIP trimer possessed slightly less complex and less extensively processed glycans, which may relate to the intrinsic stabilization as well as the absence of the membrane tether. These data provide insights into how to best use and improve membrane-associated full-length and soluble SOSIP HIV-1 Env trimers as immunogens.

The Role of Behavioral Ecotoxicology in Environmental Protection.

For decades, we have known that chemicals affect human and wildlife behavior. Moreover, due to recent technological and computational advances, scientists are now increasingly aware that a wide variety of contaminants and other environmental stressors adversely affect organismal behavior and subsequent ecological outcomes in terrestrial and aquatic ecosystems. There is also a groundswell of concern that regulatory ecotoxicology does not adequately consider behavior, primarily due to a lack of standardized toxicity methods. This has, in turn, led to the exclusion of many behavioral ecotoxicology studies from chemical risk assessments. To improve understanding of the challenges and opportunities for behavioral ecotoxicology within regulatory toxicology/risk assessment, a unique workshop with international representatives from the fields of behavioral ecology, ecotoxicology, regulatory (eco)toxicology, neurotoxicology, test standardization, and risk assessment resulted in the formation of consensus perspectives and recommendations, which promise to serve as a roadmap to advance interfaces among the basic and translational sciences, and regulatory practices.

Structure of the Lassa virus glycan shield provides a model for immunological resistance.

Lassa virus is an Old World arenavirus endemic to West Africa that causes severe hemorrhagic fever. Vaccine development has focused on the envelope glycoprotein complex (GPC) that extends from the virion envelope. The often inadequate antibody immune response elicited by both vaccine and natural infection has been, in part, attributed to the abundance of N-linked glycosylation on the GPC. Here, using a virus-like-particle system that presents Lassa virus GPC in a native-like context, we determine the composite population of each of the N-linked glycosylation sites presented on the trimeric GPC spike. Our analysis reveals the presence of underprocessed oligomannose-type glycans, which form punctuated clusters that obscure the proteinous surface of both the GP1 attachment and GP2 fusion glycoprotein subunits of the Lassa virus GPC. These oligomannose clusters are seemingly derived as a result of sterically reduced accessibility to glycan processing enzymes, and limited amino acid diversification around these sites supports their role protecting against the humoral immune response. Combined, our data provide a structure-based blueprint for understanding how glycans render the glycoprotein spikes of Lassa virus and other Old World arenaviruses immunologically resistant targets.

Sensitive Detection of SARS-CoV-2-Specific Antibodies in Dried Blood Spot Samples.

Dried blood spot (DBS) samples can be used for the detection of severe acute respiratory syndrome coronavirus 2 spike antibodies. DBS sampling is comparable to matched serum samples with a relative 98.1% sensitivity and 100% specificity. Thus, DBS sampling offers an alternative for population-wide serologic testing in the coronavirus pandemic.

SARS-CoV-2 seroprevalence and asymptomatic viral carriage in healthcare workers: a cross-sectional study.

OBJECTIVE: To determine the rates of asymptomatic viral carriage and seroprevalence of SARS-CoV-2 antibodies in healthcare workers. DESIGN: A cross-sectional study of asymptomatic healthcare workers undertaken on 24/25 April 2020. SETTING: University Hospitals Birmingham NHS Foundation Trust (UHBFT), UK. PARTICIPANTS: 545 asymptomatic healthcare workers were recruited while at work. Participants were invited to participate via the UHBFT social media. Exclusion criteria included current symptoms consistent with COVID-19. No potential participants were excluded. INTERVENTION: Participants volunteered a nasopharyngeal swab and a venous blood sample that were tested for SARS-CoV-2 RNA and anti-SARS-CoV-2 spike glycoprotein antibodies, respectively. Results were interpreted in the context of prior illnesses and the hospital departments in which participants worked. MAIN OUTCOME MEASURE: Proportion of participants demonstrating infection and positive SARS-CoV-2 serology. RESULTS: The point prevalence of SARS-CoV-2 viral carriage was 2.4% (n=13/545). The overall seroprevalence of SARS-CoV-2 antibodies was 24.4% (n=126/516). Participants who reported prior symptomatic illness had higher seroprevalence (37.5% vs 17.1%, χ2=21.1034, p<0.0001) and quantitatively greater antibody responses than those who had remained asymptomatic. Seroprevalence was greatest among those working in housekeeping (34.5%), acute medicine (33.3%) and general internal medicine (30.3%), with lower rates observed in participants working in intensive care (14.8%). BAME (Black, Asian and minority ethnic) ethnicity was associated with a significantly increased risk of seropositivity (OR: 1.92, 95% CI 1.14 to 3.23, p=0.01). Working on the intensive care unit was associated with a significantly lower risk of seropositivity compared with working in other areas of the hospital (OR: 0.28, 95% CI 0.09 to 0.78, p=0.02). CONCLUSIONS AND RELEVANCE: We identify differences in the occupational risk of exposure to SARS-CoV-2 between hospital departments and confirm asymptomatic seroconversion occurs in healthcare workers. Further investigation of these observations is required to inform future infection control and occupational health practices.