The antigenic anatomy of SARS-CoV-2 receptor binding domain.

Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 µg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.

Functional Characterization and Comparison of Plasmodium falciparum Proteins as Targets of Transmission-blocking Antibodies.

Plasmodium falciparum malaria continues to evade control efforts, utilizing highly specialized sexual-stages to transmit infection between the human host and mosquito vector. In a vaccination model, antibodies directed to sexual-stage antigens, when ingested in the mosquito blood meal, can inhibit parasite growth in the midgut and consequently arrest transmission. Despite multiple datasets for the Plasmodium sexual-stage transcriptome and proteome, there have been no rational screens to identify candidate antigens for transmission-blocking vaccine (TBV) development. This study characterizes 12 proteins from across the P. falciparum sexual-stages as possible TBV targets. Recombinant proteins are heterologously expressed as full-length ectodomains in a mammalian HEK293 cell system. The proteins recapitulate native parasite epitopes as assessed by indirect fluorescence assay and a proportion exhibits immunoreactivity when tested against sera from individuals living in malaria-endemic Burkina Faso and Mali. Purified IgG generated to the mosquito-stage parasite antigen enolase demonstrates moderate inhibition of parasite development in the mosquito midgut by the ex vivo standard membrane feeding assay. The findings support the use of rational screens and comparative functional assessments in identifying proteins of the P. falciparum transmission pathway and establishing a robust pre-clinical TBV pipeline.

Convalescent plasma therapy for the treatment of patients with COVID-19: Assessment of methods available for antibody detection and their correlation with neutralising antibody levels.

INTRODUCTION: The lack of approved specific therapeutic agents to treat coronavirus disease (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to the rapid implementation of convalescent plasma therapy (CPT) trials in many countries, including the United Kingdom. Effective CPT is likely to require high titres of neutralising antibody (nAb) in convalescent donations. Understanding the relationship between functional neutralising antibodies and antibody levels to specific SARS-CoV-2 proteins in scalable assays will be crucial for the success of a large-scale collection. We assessed whether neutralising antibody titres correlated with reactivity in a range of enzyme-linked immunosorbent assays (ELISA) targeting the spike (S) protein, the main target for human immune response. METHODS: Blood samples were collected from 52 individuals with a previous laboratory-confirmed SARS-CoV-2 infection. These were assayed for SARS-CoV-2 nAbs by microneutralisation and pseudo-type assays and for antibodies by four different ELISAs. Receiver operating characteristic (ROC) analysis was used to further identify sensitivity and specificity of selected assays to identify samples containing high nAb levels. RESULTS: All samples contained SARS-CoV-2 antibodies, whereas neutralising antibody titres of greater than 1:20 were detected in 43 samples (83% of those tested) and >1:100 in 22 samples (42%). The best correlations were observed with EUROimmun immunoglobulin G (IgG) reactivity (Spearman Rho correlation coefficient 0.88; p < 0.001). Based on ROC analysis, EUROimmun would detect 60% of samples with titres of >1:100 with 100% specificity using a reactivity index of 9.1 (13/22). DISCUSSION: Robust associations between nAb titres and reactivity in several ELISA-based antibody tests demonstrate their possible utility for scaled-up production of convalescent plasma containing potentially therapeutic levels of anti-SARS-CoV-2 nAbs.

Detection of neutralising antibodies to SARS-CoV-2 to determine population exposure in Scottish blood donors between March and May 2020.

BackgroundThe progression and geographical distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the United Kingdom (UK) and elsewhere is unknown because typically only symptomatic individuals are diagnosed. We performed a serological study of blood donors in Scotland in the spring of 2020 to detect neutralising antibodies to SARS-CoV-2 as a marker of past infection and epidemic progression.AimOur objective was to determine if sera from blood bank donors can be used to track the emergence and progression of the SARS-CoV-2 epidemic.MethodsA pseudotyped SARS-CoV-2 virus microneutralisation assay was used to detect neutralising antibodies to SARS-CoV-2. The study comprised samples from 3,500 blood donors collected in Scotland between 17 March and 18 May 2020. Controls were collected from 100 donors in Scotland during 2019.ResultsAll samples collected on 17 March 2020 (n = 500) were negative in the pseudotyped SARS-CoV-2 virus microneutralisation assay. Neutralising antibodies were detected in six of 500 donors from 23 to 26 March. The number of samples containing neutralising antibodies did not significantly rise after 5-6 April until the end of the study on 18 May. We found that infections were concentrated in certain postcodes, indicating that outbreaks of infection were extremely localised. In contrast, other areas remained comparatively untouched by the epidemic.ConclusionAlthough blood donors are not representative of the overall population, we demonstrated that serosurveys of blood banks can serve as a useful tool for tracking the emergence and progression of an epidemic such as the SARS-CoV-2 outbreak.

Role of Activins in Hepcidin Regulation during Malaria.

Epidemiological observations have linked increased host iron with malaria susceptibility, and perturbed iron handling has been hypothesized to contribute to the potentially life-threatening anemia that may accompany blood-stage malaria infection. To improve our understanding of these relationships, we examined the pathways involved in regulation of the master controller of iron metabolism, the hormone hepcidin, in malaria infection. We show that hepcidin upregulation in Plasmodium berghei murine malaria infection was accompanied by changes in expression of bone morphogenetic protein (BMP)/sons of mothers against decapentaplegic (SMAD) pathway target genes, a key pathway involved in hepcidin regulation. We therefore investigated known agonists of the BMP/SMAD pathway and found that Bmp gene expression was not increased in infection. In contrast, activin B, which can signal through the BMP/SMAD pathway and has been associated with increased hepcidin during inflammation, was upregulated in the livers of Plasmodium berghei-infected mice; hepatic activin B was also upregulated at peak parasitemia during infection with Plasmodium chabaudi Concentrations of the closely related protein activin A increased in parallel with hepcidin in serum from malaria-naive volunteers infected in controlled human malaria infection (CHMI) clinical trials. However, antibody-mediated neutralization of activin activity during murine malaria infection did not affect hepcidin expression, suggesting that these proteins do not stimulate hepcidin upregulation directly. In conclusion, we present evidence that the BMP/SMAD signaling pathway is perturbed in malaria infection but that activins, although raised in malaria infection, may not have a critical role in hepcidin upregulation in this setting.

Fatal COVID-19 outcomes are associated with an antibody response targeting epitopes shared with endemic coronaviruses.

The role of immune responses to previously seen endemic coronavirus epitopes in severe acute respiratory coronavirus 2 (SARS-CoV-2) infection and disease progression has not yet been determined. Here, we show that a key characteristic of fatal outcomes with coronavirus disease 2019 (COVID-19) is that the immune response to the SARS-CoV-2 spike protein is enriched for antibodies directed against epitopes shared with endemic beta-coronaviruses and has a lower proportion of antibodies targeting the more protective variable regions of the spike. The magnitude of antibody responses to the SARS-CoV-2 full-length spike protein, its domains and subunits, and the SARS-CoV-2 nucleocapsid also correlated strongly with responses to the endemic beta-coronavirus spike proteins in individuals admitted to an intensive care unit (ICU) with fatal COVID-19 outcomes, but not in individuals with nonfatal outcomes. This correlation was found to be due to the antibody response directed at the S2 subunit of the SARS-CoV-2 spike protein, which has the highest degree of conservation between the beta-coronavirus spike proteins. Intriguingly, antibody responses to the less cross-reactive SARS-CoV-2 nucleocapsid were not significantly different in individuals who were admitted to an ICU with fatal and nonfatal outcomes, suggesting an antibody profile in individuals with fatal outcomes consistent with an "original antigenic sin" type response.

γ-TuRC Heterogeneity Revealed by Analysis of Mozart1.

Microtubules are essential for various cell processes [1] and are nucleated by multi-protein γ-tubulin ring complexes (γ-TuRCs) at various microtubule organizing centers (MTOCs), including centrosomes [2-6]. Recruitment of γ-TuRCs to different MTOCs at different times influences microtubule array formation, but how this is regulated remains an open question. It also remains unclear whether all γ-TuRCs within the same organism have the same composition and how any potential heterogeneity might influence γ-TuRC recruitment. MOZART1 (Mzt1) was recently identified as a γ-TuRC component [7, 8] and is conserved in nearly all eukaryotes [6, 9]. Mzt1 has so far been studied in cultured human cells, yeast, and plants; its absence leads to failures in γ-TuRC recruitment and cell division, resulting in cell death [7, 9-15]. Mzt1 is small (∼8.5 kDa), binds directly to core γ-TuRC components [9, 10, 14, 15], and appears to mediate the interaction between γ-TuRCs and proteins that tether γ-TuRCs to MTOCs [9, 15]. Here, we use Drosophila to investigate the function of Mzt1 in a multicellular animal for the first time. Surprisingly, we find that Drosophila Mzt1 is expressed only in the testes and is present in γ-TuRCs recruited to basal bodies, but not to mitochondria, in developing sperm cells. mzt1 mutants are viable but have defects in basal body positioning and γ-TuRC recruitment to centriole adjuncts; sperm formation is affected and mutants display a rapid age-dependent decline in sperm motility and male fertility. Our results reveal that tissue-specific and MTOC-specific γ-TuRC heterogeneity exist in Drosophila and highlight the complexity of γ-TuRC recruitment in a multicellular animal.

Effect of anthelmintic treatment strategy on strongylid nematode species composition in grazing lambs in Scotland.

BACKGROUND: Refugia based anthelmintic protocols aim to reduce the rate of development of anthelmintic resistance in gastrointestinal nematodes (GIN). Previous studies have illustrated the impact of different drenching regimes on drug efficacy and animal growth; however, the impact on nematode populations has yet to be characterised within natural infections. This study investigated the changes in species composition of GIN throughout the grazing season, following implementation of four different ivermectin drenching regimes over six years: neo-suppressive monthly treatment (NST), targeted selective treatment (TST), strategic prophylactic treatment (SPT) and treatment upon observation of clinical signs (MT). METHODS: Lambs were grazed on one of eight replicate paddocks each grazing season following treatment regimes assigned in year 1. Faecal samples were collected fortnightly from all animals and hatched to first stage larvae (L1). DNA was extracted from individual L1 and a multiplex PCR assay targeting the internal transcribed spacer 2 (ITS2) region of Teladorsagia circumcincta, Trichostrongylus spp. and Haemonchus contortus conducted. Other species were identified using species-specific PCR. Worm-naïve tracer lambs were grazed on the paddocks at the start and end of each grazing season and adult worms recovered at post mortem to investigate the parasite population on pasture. RESULTS: Results showed an overall decrease in species diversity in egg output from the NST group which occurred within a single grazing season and was consistent throughout the experiment. Species diversity was protected over six years in groups implementing TST, SPT and MT treatment regimes, designed to offer refugia. The expected shift in species prevalence throughout the season from Teladorsagia to Trichostrongylus was observed in all but the NST group where only Teladorsagia spp. were recovered from trial lambs by the end of the experiment. Worm burdens indicated the presence of several species at relatively low abundance on pasture in the NST group in 2011. However, these species were not represented in egg output from trial lambs, probably due to the frequent anthelmintic treatment administered throughout the grazing season. CONCLUSION: The molecular methods utilised here worked well. The comparable results of the three refugia-based treatment regimes suggest that nematode diversity can be maintained using part or whole group treatments if a rich supra-population of parasites are available to re-infect animals post treatment.

Enhancing immunogenicity and transmission-blocking activity of malaria vaccines by fusing Pfs25 to IMX313 multimerization technology.

Transmission-blocking vaccines (TBV) target the sexual-stages of the malaria parasite in the mosquito midgut and are widely considered to be an essential tool for malaria elimination. High-titer functional antibodies are required against target antigens to achieve effective transmission-blocking activity. We have fused Pfs25, the leading malaria TBV candidate antigen to IMX313, a molecular adjuvant and expressed it both in ChAd63 and MVA viral vectors and as a secreted protein-nanoparticle. Pfs25-IMX313 expressed from viral vectors or as a protein-nanoparticle is significantly more immunogenic and gives significantly better transmission-reducing activity than monomeric Pfs25. In addition, we demonstrate that the Pfs25-IMX313 protein-nanoparticle leads to a qualitatively improved antibody response in comparison to soluble Pfs25, as well as to significantly higher germinal centre (GC) responses. These results demonstrate that antigen multimerization using IMX313 is a very promising strategy to enhance antibody responses against Pfs25, and that Pfs25-IMX313 is a highly promising TBV candidate vaccine.