Brewpitopes: a pipeline to refine B-cell epitope predictions during public health emergencies (preprint)

The application of B-cell epitope identification for the development of therapeutic antibodies is well established but consuming in terms of time and resources. For this reason, in the last few years, the immunoinformatic community has developed several computational predictive tools. While relatively successful, most of these tools only use a few properties of the candidate region to determine their likelihood of being a true B-cell epitope. However, this likelihood is influenced by a wide variety of protein features, including the presence of glycosylated residues in the neighbourhood of the candidate epitope, the subcellular location of the protein region or the three-dimensional information about their surface accessibility in the parental protein. In this study we created Brewpitopes, an integrative pipeline to curate computational predictions of B-cell epitopes by accounting for all the aforementioned features. To this end, we implemented a set of rational filters to mimic the conditions for the in vivo antibody recognition to enrich the B-cell epitope predictions in actionable candidates. To validate Brewpitopes, we analyzed the SARS-CoV-2 proteome. In the S protein, Brewpitopes enriched the initial predictions in 5-fold on epitopes with neutralizing potential (p-value < 2e-4). Other than S protein, 4 out of 16 proteins in the proteome contain curated B-cell epitopes and hence, have also potential interest for viral neutralization, since mutational escape mainly affects the S protein. Our results demonstrate that Brewpitopes is a powerful pipeline for the rapid prediction of refined B-cell epitopes during public health emergencies.

ICU-Acquired Pneumonia is a Risk Factor of a Poor Health Post-Covid-19 Syndrome (preprint)

Background: . Some patients who had previously presented with COVID-19 have been reported to develop persistent COVID-19 symptoms. Whilst this information has been adequately recognised and extensively published with respect to non-critically ill patients, less is known about the prevalence and risk factors and characteristics of persistent COVID_19 . On other hand these patients have very often intensive care unit-acquired pneumonia (ICUAP). A second infectious hit after COVID increases the length of ICU stay and mechanical ventilation and could have an influence in the poor health post-Covid 19 syndrome in ICU discharged patients Methods: This prospective, multicentre and observational study was done across 40 selected ICUs in Spain. Consecutive patients with COVID-19 requiring ICU admission were recruited and evaluated three months after hospital discharge. Results: A total of 1,255 ICU patients were scheduled to be followed up at 3 months; however, the final cohort comprised 991 (78.9%) patients. A total of 315 patients developed ICUAP (97% of them had ventilated ICUAP) Patients requiring invasive mechanical ventilation had persistent, post-COVID-19 symptoms than those who did not require mechanical ventilation. Female sex, duration of ICU stay, and development of ICUAP were independent risk factors for persistent poor health post-COVID-19. Conclusions: : Persistent, post-COVID-19 symptoms occurred in more than two-thirds of patients. Female sex, duration of ICU stay and the onset of ICUAP comprised all independent risk factors for persistent poor health post-COVID-19. Prevention of ICUAP could have beneficial effects in poor health post-Covid 19

Label-Free Plasmonic Biosensor for Rapid, Quantitative, and Highly Sensitive COVID-19 Serology: Implementation and Clinical Validation (preprint)

Serological tests are essential for the control and management of COVID-19 pandemic, not only for current and historical diagnostics but especially for surveillance, epidemiological, and acquired immunity studies. Clinical COVID-19 serology is routinely performed by enzymatic or chemiluminescence immunoassays (i.e., ELISA or CLIA), which provide good sensitivities at the expense of relatively long turnaround times and specialized laboratory settings. Rapid serological tests, based on lateral flow assays, have also been developed and widely commercialized, but they suffer from limited reliability due to relatively low sensitivity and specificity. We have developed and validated a direct serological biosensor assay employing proprietary technology based on Surface Plasmon Resonance (SPR). The biosensor offers a rapid -less than 15 min- identification and quantification of SARS-CoV-2 antibodies directly in clinical samples, without the need of any signal amplification. The portable plasmonic biosensor device employs a custom-designed multi-antigen sensor biochip, combining the two main viral antigens (RBD peptide and N protein), for simultaneous detection of human antibodies targeting both antigens. The SPR serology assay reaches detection limits in the low ng mL-1 range employing polyclonal antibodies as standard, which are well below the commonly detected antibody levels in COVID-19 patients. The assay has also been implemented employing the first WHO approved anti-SARS-CoV-2 immunoglobulin standard. We have carried out a clinical validation with COVID-19 positive and negative samples (n=120) that demonstrates the excellent diagnostic sensitivity (99%) and specificity (100%). This positions our biosensor device as an accurate, robust, and easy-to-use diagnostics tool for rapid and reliable COVID-19 serology to be employed both at laboratory and decentralized settings for the management of COVID-19 patients and for the evaluation of immunological status during vaccination, treatment or in front of emerging variants.

Absent or insufficient anti-SARS-CoV-2 S antibodies at ICU admission are associated to higher viral loads in plasma, antigenemia and mortality in COVID-19 patients (preprint)

Purposeto evaluate the association between anti-SARS-CoV-2 S IgM and IgG antibodies with viral RNA load in plasma, the frequency of antigenemia and with the risk of mortality in critically ill patients with COVID-19. Methodsanti-SARS-CoV-2 S antibodies levels, viral RNA load and antigenemia were profiled in plasma of 92 adult patients in the first 24 hours following ICU admission. The impact of these variables on 30-day mortality was assessed by using Kaplan-Meier curves and multivariate Cox regression analysis. Resultsnon survivors showed more frequently absence of anti-SARS-CoV-2 S IgG and IgM antibodies than survivors (26.3% vs 5.6% for IgM and 18.4% vs 5.6% for IgG), and a higher frequency of antigenemia (47.4% vs 22.2%) (p <0.05). Non survivors showed lower concentrations of anti-S IgG and IgM and higher viral RNA loads in plasma, which were associated to increased 30-day mortality and decreased survival mean time. [Adjusted HR (CI95%), p]: [S IgM (AUC [≥]60): 0.48 (0.24; 0.97), 0.040]; [S IgG (AUC [≥]237): 0.47 (0.23; 0.97), 0.042]; [Antigenemia (+): 2.45 (1.27; 4.71), 0.007]; [N1 viral load ([≥] 2.156 copies/mL): 2.21 (1.11; 4.39),0.024]; [N2 viral load ([≥] 3.035 copies/mL): 2.32 (1.16; 4.63), 0.017]. Frequency of antigenemia was >2.5-fold higher in patients with absence of antibodies. Levels of anti-SARS-CoV-2 S antibodies correlated inversely with viral RNA load. Conclusionabsence / insufficient levels of anti-SARS-CoV-2 S antibodies following ICU admission is associated to poor viral control, evidenced by increased viral RNA loads in plasma, higher frequency of antigenemia, and also to increased 30-day mortality. Take-home messageabsent or low levels of antibodies against the S protein of SARS-CoV- 2 at ICU admission is associated to an increased risk of mortality, higher frequency of antigenemia and higher viral RNA loads in plasma. Profiling anti-SARS-CoV-2 s antibodies at ICU admission could help to predict outcome and to better identify those patients potentially deserving replacement treatment with monoclonal or polyclonal antibodies.

Immunogenicity and crossreactivity of antibodies to SARS-CoV-2 nucleocapsid protein (preprint)

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 indentified a C-terminus region in SARS-CoV2 N of minimal sequence homology with HCoV that was more specific and highly immunogenic. IgGs to the full-length SARS-CoV-2 N also recognised N229E N, and IgGs to HKU1 N recognised 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 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 symptoms and in females. IgGs remained stable for at least 3 months, while IgAs and IgMs declined faster. In conclusion, N 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.

SARS-CoV-2-RNA viremia is associated to hypercytokinemia and critical illness in COVID-19 (preprint)

BackgroundSevere COVID-19 is characterized by clinical and biological manifestations typically observed in sepsis. SARS-CoV-2 RNA is commonly detected in nasopharyngeal swabs, however viral RNA can be found also in peripheral blood and other tissues. Whether systemic spreading of the virus or viral components plays a role in the pathogenesis of the sepsis-like disease observed in severe COVID-19 is currently unknown. MethodsWe determined the association of plasma SARS-CoV-2 RNA with the biological responses and the clinical severity of patients with COVID-19. 250 patients with confirmed COVID-19 infection were recruited (50 outpatients, 100 hospitalised ward patients, and 100 critically ill). The association between plasma SARS-CoV-2 RNA and laboratory parameters was evaluated using multivariate GLM with a gamma distribution. The association between plasma SARS-CoV-2 RNA and severity was evaluated using multivariate ordinal logistic regression analysis and Generalized Linear Model (GLM) analysis with a binomial distribution. ResultsThe presence of SARS-CoV-2-RNA viremia was independently associated with a number of features consistently identified in sepsis: 1) high levels of cytokines (including CXCL10, CCL-2, IL-10, IL-1ra, IL-15, and G-CSF); 2) higher levels of ferritin and LDH; 3) low lymphocyte and monocyte counts 4) and low platelet counts. In hospitalised patients, the presence of SARS-CoV-2-RNA viremia was independently associated with critical illness: (adjusted OR= 8.30 [CI95%=4.21 - 16.34], p < 0.001). CXCL10 was the most accurate identifier of SARS-CoV-2-RNA viremia in plasma (area under the curve (AUC), [CI95%], p) = 0.85 [0.80 - 0.89), <0.001]), suggesting its potential role as a surrogate biomarker of viremia. The cytokine IL-15 most accurately differentiated clinical ward patients from ICU patients (AUC: 0.82 [0.76 - 0.88], <0.001). Conclusionssystemic dissemination of genomic material of SARS-CoV-2 is associated with a sepsis-like biological response and critical illness in patients with COVID-19. RNA viremia could represent an important link between SARS-CoV-2 infection, host response dysfunction and the transition from moderate illness to severe, sepsis-like COVID-19 disease.