Unraveling the long-term kinetics of antibodies to SARS-CoV-2 and the individual characteristics influencing it, including the impact of pre-existing antibodies to human coronaviruses causing common cold (HCoVs), is essential to understand protective immunity to COVID-19 and devise effective surveillance strategies. IgM, IgA and IgG levels against six SARS-CoV-2 antigens and the nucleocapsid antigen of the four HCoV (229E, NL63, OC43 and HKU1) were quantified by Luminex, and antibody neutralization capacity was assessed by flow cytometry, in a cohort of health care workers followed up to 7 months (N = 578). Seroprevalence increases over time from 13.5% (month 0) and 15.6% (month 1) to 16.4% (month 6). Levels of antibodies, including those with neutralizing capacity, are stable over time, except IgG to nucleocapsid antigen and IgM levels that wane. After the peak response, anti-spike antibody levels increase from ~150 days post-symptom onset in all individuals (73% for IgG), in the absence of any evidence of re-exposure. IgG and IgA to HCoV are significantly higher in asymptomatic than symptomatic seropositive individuals. Thus, pre-existing cross-reactive HCoVs antibodies could have a protective effect against SARS-CoV-2 infection and COVID-19 disease.

Reliable serological tests are required to determine the prevalence of antibodies against SARS-CoV-2 and to characterise immunity to the disease in order to address key knowledge gaps in the COVID-19 pandemic. Quantitative suspension array technology (qSAT) assays based on the xMAP Luminex platform overcome the limitations of rapid diagnostic tests and ELISA with their higher precision, dynamic range, throughput, miniaturization, cost-efficacy and multiplexing capacity. We developed three qSAT assays for IgM, IgA and IgG to a panel of eight SARS-CoV-2 antigens including spike (S), nucleoprotein (N) and membrane (M) protein constructs. The assays were optimized to minimize processing time and maximize signal to noise ratio. We evaluated their performance using 128 pre-pandemic plasmas (negative controls) and 104 plasmas from individuals with SARS-CoV-2 diagnosis (positive controls), of whom 5 were asymptomatic, 51 had mild symptoms and 48 were hospitalized. Pre-existing IgG antibodies recognizing N, M and S proteins were detected in negative controls suggestive of cross-reactive to common cold coronaviruses. The best performing antibody/antigen signatures had specificities of 100% and sensitivities of 95.78% at ≥14 days and 95.65% at ≥21 days since the onset of symptoms, with AUC of 0.977 and 0.999, respectively. Combining multiple markers as assessed by qSAT assays has the highest efficiency, breadth and versatility to accurately detect low-level antibody responses for obtaining reliable data on prevalence of exposure to novel pathogens in a population. Our assays will allow gaining insights into antibody correlates of immunity and their kinetics, required for vaccine development to combat the COVID-19 pandemic.

COVID-19 patients elicit strong responses to the nucleocapsid (N) protein of SARS-CoV-2 but binding antibodies are also detected in prepandemic individuals, indicating potential crossreactivity with common cold human coronaviruses (HCoV) and questioning its utility in seroprevalence studies. We investigated the immunogenicity of the full-length and shorter fragments of the SARS-CoV-2 N protein, and the crossreactivity of antibodies with HCoV. We identified a C-terminus region in SARS-CoV2 N of minimal sequence homology with HCoV that was more specific for SARS-CoV-2 and highly immunogenic. IgGs to the full-length SARS-CoV-2 N also recognized N229E N, and IgGs to HKU1 N recognized SARS-CoV-2 N. Crossreactivity with SARS-CoV-2 was stronger for alpha-rather than beta-HCoV despite having less sequence identity, revealing the importance of conformational recognition. Higher preexisting IgG to OC43 N correlated with lower IgG to SARS-CoV-2 N in rRT-PCR negative individuals, reflecting less exposure and indicating a potential protective association. Antibodies to SARS-CoV-2 N were higher in patients with more severe and longer duration of symptoms and in females. IgGs remained stable for at least 3 months, while IgAs and IgMs declined faster. In conclusion, N protein is a primary target of SARS-CoV-2-specific and HCoV crossreactive antibodies, both of which may affect the acquisition of immunity to COVID-19.

The blood-brain barrier (BBB) is a formidable obstacle for brain delivery of therapeutic antibodies. However, antibodies against the transferrin receptor (TfR), enriched in brain endothelial cells, have been developed as delivery carriers of therapeutic cargoes into the brain via a receptor-mediated transcytosis pathway. In vitro and in vivo studies demonstrated that either a low-affinity or monovalent binding of these antibodies to the TfR improves their release on the abluminal side of the BBB and target engagement in brain parenchyma. However, these studies have been performed with mouse-selective TfR antibodies that recognize different TfR epitopes and have varied binding characteristics. In this study, we evaluated serum pharmacokinetics and brain and CSF exposure of the rat TfR-binding antibody OX26 affinity variants, having Ks of 5 nM, 76 nM, 108 nM, and 174 nM, all binding the same epitope in bivalent format. Pharmacodynamic responses were tested in the Hargreaves chronic pain model after conjugation of OX26 affinity variants with the analgesic and antiepileptic peptide, galanin. OX26 variants with affinities of 76 nM and 108 nM showed enhanced brain and cerebrospinal fluid (CSF) exposure and higher potency in the Hargreaves model, compared to a 5 nM affinity variant; lowering affinity to 174 nM resulted in prolonged serum pharmacokinetics, but reduced brain and CSF exposure. The study demonstrates that binding affinity optimization of TfR-binding antibodies could improve their brain and CSF exposure even in the absence of monovalent TfR engagement.

Protein modification by SUMO modulates essential biological processes in eukaryotes. SUMOylation is facilitated by sequential action of the E1-activating, E2-conjugating, and E3-ligase enzymes. In plants, SUMO regulates plant development and stress responses, which are key determinants in agricultural productivity. To generate additional tools for advancing our knowledge about the SUMO biology, we have developed a strategy for inhibiting in vivo SUMO conjugation based on disruption of SUMO E1-E2 interactions through expression of E1 SAE2 domain. Targeted mutagenesis and phylogenetic analyses revealed that this inhibition involves a short motif in SAE2 highly divergent across kingdoms. Transgenic plants expressing the SAE2 domain displayed dose-dependent inhibition of SUMO conjugation, and have revealed the existence of a post-transcriptional mechanism that regulates SUMO E2 conjugating enzyme levels. Interestingly, these transgenic plants displayed increased susceptibility to necrotrophic fungal infections by Botrytis cinerea and Plectosphaerella cucumerina. Early after fungal inoculation, host SUMO conjugation was post-transcriptionally downregulated, suggesting that targeting SUMOylation machinery could constitute a novel mechanism for fungal pathogenicity. These findings support the role of SUMOylation as a mechanism involved in plant protection from environmental stresses. In addition, the strategy for inhibiting SUMO conjugation in vivo described in this study might be applicable in important crop plants and other non-plant organisms regardless of their genetic complexity.