Neutralizing antibodies to Omicron after the fourth SARS-CoV-2 mRNA vaccine dose in immunocompromised patients highlight the need of additional boosters (preprint)

Immunocompromised patients have been shown to have an impaired immune response to COVID-19 vaccines. Here we compared the B-cell, T-cell and neutralizing antibody response to WT and Omicron BA.2 SARS-CoV-2 virus after the fourth dose of mRNA COVID-19 vaccines in patients with hematological malignancies (HM, n=71), solid tumors (ST, n=39) and immune-rheumatological (ID, n=25) diseases. We show that the T-cell response is similarly boosted by the fourth dose across the different subgroups, while the antibody response is improved only in patients not receiving B-cell targeted therapies, independent on the pathology. However, 9% of patients with anti-RBD antibodies did not have neutralizing antibodies to both virus variants, while an additional 5.7% did not have neutralizing antibodies to Omicron BA.2, making these patients particularly vulnerable to SARS-CoV-2 infection. The increment of neutralizing antibodies was very similar towards Omicron BA.2 and WT virus after the third or fourth dose of vaccine, suggesting that there is no preferential skewing towards either virus variant with the booster dose. The only limited step is the amount of antibodies that are elicited after vaccination, thus increasing the probability of developing neutralizing antibodies to both variants of virus. Hence, additional booster doses are recommended to frail patients.

Factors Modulating COVID-19: A Mechanistic Understanding Based on the Adverse Outcome Pathway Framework (preprint)

Addressing factors modulating COVID-19 is crucial since abundant clinical evidence shows that outcomes are markedly heterogeneous between patients. This requires identifying the factors and understanding how they mechanistically influence COVID-19. Here, we describe how eleven selected factors influence COVID-19 by applying the Adverse Outcome Pathway (AOP) framework well-established in regulatory toxicology. This framework aims to model the sequence of events starting from an initial interaction of a stressor with the organism and the progress through key biological events leading to an adverse health outcome. Several linear AOPs depicting pathways from the binding of the virus to ACE2 up to clinical outcomes observed in COVID-19 patients have been developed and integrated into a network offering a unique overview of the mechanisms underlying the disease. As SARS-CoV-2 infectibility and ACE2 activity are the major starting points and inflammatory response is central in the development of COVID-19, we evaluated how eleven intrinsic and extrinsic factors modulate those processes impacting clinical outcomes. Applying this AOP-aligned approach enables the identification of current knowledge gaps orientating for further research and allows to propose biomarkers to identify of high-risk patients. This approach also facilitates expertise synergy from different disciplines to address public health issues.

mRNA-COVID19 vaccination can be considered safe and tolerable for frail patients (preprint)

Background Frail patients are considered at relevant risk of complications due to COVID-19 infection and, for this reason, are prioritized candidates for vaccination. As these patients were originally not included in the registration trials, fear related to vaccine side-effects and disease worsening was one of the reasons for vaccine hesitancy. Herein we report the safety profile of the prospective, multicenter, national VAX4FRAIL study (NCT04848493) to evaluate vaccines in a large trans-disease cohort of patients with solid or hematological malignancies, neurological and rheumatological diseases. Methods Between March 3rd and September 2nd, 2021, 566 patients were evaluable for safety endpoint: 105 received the mRNA-1273 vaccine and 461 the BNT162b2 vaccine. Frail patients were defined per protocol as patients under treatment with hematological malignancies (131), solid tumors (191), immune-rheumatological diseases (86), and neurological diseases (158), including multiple sclerosis and generalized myasthenia. The impact of the vaccination on the health status of patients was assessed through a questionnaire focused on the first week after each vaccine dose. Results The most frequently reported moderate-severe adverse events were pain at the injection site (60.3% after the first dose, 55.4% after the second), fatigue (30.1% - 41.7%), bone pain (27.4% - 27.2%) and headache (11.8% - 18.9%). Risk factors associated with the occurrence of severe symptoms after vaccine administration were identified through a multivariate logistic regression analysis: age was associated with severe fever presentation (younger patients vs. middle-aged vs. older ones), females presented a higher probability of severe pain at the injection site, fatigue, headache, and bone pain; the mRNA-1237 vaccine was associated with a higher probability of severe pain at the injection site and fever. After the first dose, patients presenting a severe symptom were at a relevant risk of recurrence of the same severe symptom after the second one. Overall, 11 patients (1.9%) after the first dose and 7 (1.2%) after the second one required to postpone or suspend the disease-specific treatment. Finally, 2 fatal events occurred among our 566 patients. These two events were considered unrelated to the vaccine. Conclusions Our study reports that mRNA-COVID-19 vaccination is safe also in frail patients as expected side effects were manageable and had a minimum impact on patient care path.

mRNA COVID-19 vaccine booster fosters B and T cell responses in immunocompromised patients (preprint)

SARS-CoV-2 vaccination has proven effective in inducing an immune response in healthy individuals and is progressively allowing to overcome the pandemic. Recent evidence has shown that response to vaccination in some vulnerable patients may be diminished, and it has been proposed a booster dose. We tested the kinetic of development of serum antibodies to the SARS-CoV-2 Spike protein, their neutralizing capacity, the CD4 and CD8 IFN-γ T cell response in 328 subjects, including 131 immunocompromised individuals (cancer, rheumatologic, and hemodialysis patients), 160 healthcare workers (HCW) and 37 subjects older than 75 yo, after vaccination with two or three doses of mRNA vaccines. We stratified the patients according to the type of treatment. We found that immunocompromised patients, depending on the type of treatment, poorly respond to SARS-CoV-2 mRNA vaccines. However, an additional booster dose of vaccine induced a good immune response in almost all of the patients except those receiving anti-CD20 antibody. Similarly to HCW, previously infected and vaccinated immunocompromised individuals demonstrate a stronger SARS-CoV-2 specific immune response than those who are vaccinated without prior infection. Summary blurb Immunocompromised patients poorly respond to two doses of SARS-CoV-2 mRNA vaccines. However, an additional booster dose elicits a strong humoral and cellular immune response in these subjects.

Humoral and T-cell immune response after three doses of mRNA SARS-CoV-2 vaccines in fragile patients: the Italian VAX4FRAIL study (preprint)

BackgroundPatients with solid or hematological tumors, neurological and immune-inflammatory disorders represent potentially fragile subjects with increased risk to experience severe COVID-19 and inadequate response to SARS-CoV2 vaccination. MethodsWe designed a prospective Italian multicentric study to assess humoral and T-cell response to SARS-CoV2 vaccination in patients (n=378) with solid tumors (ST), hematological malignancies (HM), neurological (ND) and immuno-rheumatological diseases (ID). The immunogenicity of primary vaccination schedule and of the booster dose were analyzed. ResultsOverall, patient seroconversion rate after two doses was 62.1%. A significant lower rate was observed in HM (52.4%) and ID (51.9%) patients compared to ST (95.6%) and ND (70.7%); a lower median level of antibodies was detected in HM and ID versus the others (p<0.0001). A similar rate of patients with a positive SARS-CoV2 T-cell response was observed in all disease groups, with a higher level observed in the ND group. The booster dose improved humoral responses in all disease groups, although with a lower response in HM patients, while the T-cell response increased similarly in all groups. In the multivariable logistic model, the independent predictors for seroconversion were disease subgroups, type of therapies and age. Notably, the ongoing treatment known to affect the immune system was associated with the worst humoral response to vaccination (p<0.0001), but had no effects on the T-cell responses. ConclusionsImmunosuppressive treatment more than disease type per se is a risk factor for low humoral response after vaccination. The booster dose can improve both humoral and T-cell response. Articles main point- Lower rate of seroconversion was observed in fragile patients as compared to healthy controls - The booster dose improves humoral and T-cell response in all fragile patient groups - Immunosuppressive treatment was associated with the worst humoral response to vaccination, but had no effects on T-cell responses.

A PTX3/LDH/CRP signature correlates with lung injury CTs scan severity and disease progression in paucisymptomatic COVID-19 (preprint)

BackgroundQuantitative CT (QCT) analysis is an invaluable diagnostic tool to assess lung injury and predict prognosis of patients affected by COVID-19 pneumonia. PTX3 was recently described as one of the most reliable serological predictors of clinical deterioration and short-term mortality. The present study was designed to evaluate a correlation between serological biomarkers of inflammation and lung injury measured by QCT. MethodsThis retrospective monocentric study analysed a cohort of patients diagnosed with COVID-19 and admitted because of respiratory failure, or significant radiological involvement on chest CT scan. All patients, males and non-pregnant females older than 18 years, underwent chest CT scan and laboratory testing at admission. Exclusion criteria were defined by concurrent acute pathological processes and ongoing specific treatments which could interfere with immune activity. The cohort was stratified based on severity in mild and severe forms. Compromised lung at QCT was then correlated to serological biomarkers representative of SARS-CoV-2. We further developed a multivariable logistic model to predict CT data and clinical deterioration based on a specific molecular signature. Internal cross-validation led to evaluate discrimination, calibration, and clinical utility of the tool that was provided by a score to simplify its application. Findings592 patients were recruited between March 19th and December 1st, 2020. Applying exclusion criteria which consider confounders, the cohort resulted in 366 individuals characterized by 177 mild and 189 severe forms. In our predictive model, blood levels of PTX3, CRP and LDH were found to correlate with QCT values in mild COVID-19 disease. A signature of these three biomarkers had a high predictive accuracy in detecting compromised lungs as assessed by QCT. The score was elaborated and resulted representative of lung CT damage leading to clinical deterioration and oxygen need in mild disease. InterpretationThe LDH, PTX3, CRP blood signature can serve as a strong correlate of compromised lung in COVID-19, possibly integrating cellular damage, systemic inflammation, myeloid and endothelial cell activation. FundingThis work was supported by a philanthropic donation by Dolce & Gabbana fashion house (to A.M., C.G.) and by a grant from Italian Ministry of Health for COVID-19 (to A.M. and C.G.). Research in contextO_ST_ABSEvidence before this studyC_ST_ABSBesides nasopharyngeal swab and serological test, chest CT scan represents one of the most useful tools to confirm COVID-19 diagnosis; moreover, QCT has been demonstrated to foresee oxygen need as well as deterioration of health status. Several clinical and serological parameters have been studied alone or combined in scores to be applied as prognostic tools of SARS-CoV-2 pneumonia; however, no one has yet reached the everyday practice. Recently, our group has investigated the expression and clinical significance of PTX3 in COVID-19 demonstrating the correlation with short-term mortality independently of confounders. The result was confirmed by other studies in different settings increasing evidence of PTX3 as a strong biomarker of severity; noteworthy, a recent report analysed proteomic data with a machine learning approach identifying age with PTX3 or SARS-CoV-2 RNAemia as the best binary signatures associated to 28-days mortality. Added value of this studyThe present study was designed to investigate associations between markers of damage and the CT extension of SARS-CoV-2 pneumonia in order to provide a biological footprint of radiological results in paucisymptomatic patients. QCT data were considered in a binary form identifying a threshold relevant for clinical deterioration, as already proved by literature. Our findings demonstrate a significant correlation with three peripheral blood proteins (PTX3, LDH and CRP) which result representative of COVID-19 severity. The study presents a predictive model of radiological lung involvement which performs with a high level of accuracy (cvAUC of 0{middle dot}794{+/-}0{middle dot}107; CI 95%: 0{middle dot}74-0{middle dot}87) and a simple score was provided to simplify the interpretation of the three biomarkers. Besides additional finding on PTX3 role in SARS-CoV2 pathology, its prognostic value was confirmed by data on clinical deterioration; indeed, paucisymptomatic subjects showed a 11{middle dot}9% deaths. The model offers the possibility to quickly assess patients resulted positive for SARS-CoV-2 and estimate people at risk of deterioration despite normal clinical and blood gases analysis, with potential to identify those who need better clinical monitoring and interventions. Implications of all the available evidencePredicting the extension, severity, and clinical deterioration in COVID-19 patients its pivotal to allocate enough resources in emergency and to avoid health system burden. Despite the urgent clinical need of biomarkers, SARS-CoV-2 pneumonia still lacks something able to provide an easy measure of its severity. Some multiparametric scores have been proposed for severe COVID-19 and rely on deep assessment of patients status (clinical, serological, and radiological data). Our model represents an unprecedented effort to provide a tool which could predict CT pneumonia extension, oxygen requirement and clinical deterioration in mild COVID-19. Based on the measurement of three proteins on peripheral blood, this score could improve early assessment of asymptomatic patients tested positive by SARS-CoV2 specifically in first level hospitals as well in developing countries.

New susceptibility loci for severe COVID-19 by detailed GWAS analysis in European populations (preprint)

Due to the highly variable clinical phenotype of Coronavirus disease 2019 (COVID-19), deepening the host genetic contribution to severe COVID-19 may further improve our understanding about underlying disease mechanisms. Here, we describe an extended GWAS meta-analysis of 3,260 COVID-19 patients with respiratory failure and 12,483 population controls from Italy, Spain, Norway and Germany, as well as hypothesis-driven targeted analysis of the human leukocyte antigen (HLA) region and chromosome Y haplotypes. We include detailed stratified analyses based on age, sex and disease severity. In addition to already established risk loci, our data identify and replicate two genome-wide significant loci at 17q21.31 and 19q13.33 associated with severe COVID-19 with respiratory failure. These associations implicate a highly pleiotropic ~0.9-Mb 17q21.31 inversion polymorphism, which affects lung function and immune and blood cell counts, and the NAPSA gene, involved in lung surfactant protein production, in COVID-19 pathogenesis.

Recognition and inhibition of SARS-CoV-2 by humoral innate immunity pattern recognition molecules (preprint)

Summary The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in COVID-19. The present study was designed to conduct a systematic investigation of the interaction of humoral fluid phase pattern recognition molecules (PRM) with SARS-CoV-2. Out of 10 PRM tested, the long pentraxin PTX3 and Mannose Binding Lectin (MBL) bound the viral Nucleoprotein and Spike, respectively. MBL bound trimeric Spike, including that of variants of concern, in a glycan- dependent way and inhibited SARS-CoV-2 in three in vitro models. Moreover, upon binding to Spike, MBL activated the lectin pathway of complement activation. Genetic polymorphisms at the MBL locus were associated with disease severity. These results suggest that selected humoral fluid phase PRM can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.

SARS-CoV-2-associated ssRNAs activate inflammation and immunity via TLR7/8 (preprint)

The inflammatory and IFN pathways of innate immunity play a key role in both resistance and pathogenesis of Coronavirus Disease 2019 (COVID-19). Innate sensors and SARS-CoV-2-Associated Molecular Patterns (SAMPs) remain to be completely defined. Here we identify single-stranded RNA (ssRNA) fragments from SARS-CoV-2 genome as direct activators of endosomal TLR7/8 and MyD88 pathway. The same sequences induced human DC activation in terms of phenotype and functions, such as IFN and cytokine production and Th1 polarization. A bioinformatic scan of the viral genome identified several hundreds of fragments potentially activating TLR7/8, suggesting that products of virus endosomal processing potently activate the IFN and inflammatory responses downstream these receptors. In vivo, SAMPs induced MyD88-dependent lung inflammation characterized by accumulation of proinflammatory and cytotoxic mediators and immune cell infiltration, as well as splenic DC phenotypical maturation. These results identify TLR7/8 as crucial cellular sensors of ssRNAs encoded by SARS-CoV-2 involved in host resistance and disease pathogenesis of COVID-19.

COVID-19 during pregnancy and adverse outcomes: Concerns and recommendations from The Brazilian Teratology Information Service.

SARS-CoV-2 virus was first identified in the beginning of 2020 and has spread all over the world, causing the Coronavirus Disease 2019 (COVID-19) pandemic. The virus is a member of the Coronavirus family, which includes viruses that cause common cold, Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). MERS and SARS are known by causing adverse events in pregnancy. Considering that SARS-CoV-2 is a new infection agent, little is known about the risk of its infection to human embryo/fetal development. However, SARS and MERS were associated with negative outcomes, such as miscarriage, preterm birth, intrauterine growth restriction and perinatal death. Here, we raise concerns and possibilities related the harmful potential of SARS-CoV-2 and COVID-19 to pregnancy, discussing symptoms, immunological changes during pregnancy, SARS-CoV-2 mutation rate (and the risks related to it). Finally, we point out recommendations to be performed by the scientific community and health care workers in order to identify and to manage potential risks to pregnant women and their babies.

The antibody response to SARS-CoV-2 increases over 5 months in patients with anosmia/dysgeusia (preprint)

The factors involved in the persistence of antibodies to SARS-CoV-2 are unknown. We evaluated the antibody response to SARS-CoV-2 in personnel from 10 healthcare facilities and its association with individuals' characteristics and COVID-19 symptoms in an observational study. We enrolled 4735 subjects (corresponding to 80% of all personnel) over a period of 5 months when the spreading of the virus was drastically reduced. For each participant, we determined the rate of antibody increase or decrease over time in relation to 93 features analyzed in univariate and multivariate analyses through a machine learning approach. In individuals positive for IgG (>= 12 AU/mL) at the beginning of the study, we found an increase [p= 0.0002] in antibody response in symptomatic subjects, particularly with anosmia/dysgeusia (OR 2.75, 95% CI 1.753 - 4.301), in a multivariate logistic regression analysis. This may be linked to the persistence of SARS-CoV-2 in the olfactory bulb.

A cautionary note on recall vaccination in ex-COVID-19 subjects (preprint)

Currently approved COVID-19 vaccines based on mRNA or adenovirus require a first jab followed by recall immunization. There is no indication as to whether individuals who have recovered from COVID-19 should be vaccinated, and if so, if they should receive one or two vaccine doses. Here, we tested the antibody response developed after the first dose of the mRNA based vaccine encoding the SARS-CoV-2 full-length spike protein (BNT162b2) in 124 healthcare professionals of which 57 had a previous history of COVID-19 (ExCOVID). Post-vaccine antibodies in ExCOVID individuals increase exponentially within 7-15 days after the first dose compared to naive subjects (p<0.0001). We developed a multivariate Linear Regression (LR) model with l2 regularization to predict the IgG response for SARS-COV-2 vaccine. We found that the antibody response of ExCOVID patients depends on the IgG pre-vaccine titer and on the symptoms that they developed during the disorder, with anosmia/dysgeusia and gastrointestinal disorders being the most significantly positively correlated in the LR. Thus, one vaccine dose is sufficient to induce a good antibody response in ExCOVID subjects. This poses caution for ExCOVID subjects to receive a second jab both because they may have a overreaction of the inflammatory response and also in light of the current vaccine shortage.

Macropahge expression and prognostic significance of the long pentraxin PTX3 in COVID-19 (preprint)

PTX3 is an essential component of humoral innate immunity, involved in resistance to selected pathogens and in the regulation of inflammation. PTX3 plasma levels are associated with poor outcome in systemic inflammatory conditions and vascular pathology. The present study was designed to assess expression and significance of PTX3 in COVID-19. By bioinformatics analysis of public databases PTX3 expression was detected in lung respiratory cell lines exposed to SARS-CoV-2. By analysis at single cell level of COVID-19 circulating mononuclear cells, we found that PTX3 was selectively expressed by monocytes among circulating leukocytes. Moreover, in lung bronchoalveolar lavage fluid, single cell analysis revealed selective expression of PTX3 in neutrophils and macrophages, which play a major role in the pathogenesis of the disease. By immunohistochemistry, PTX3 was expressed by lung myelomocytic cells, type 2 pneumocytes and vascular endothelial cells. PTX3 plasma levels were determined by ELISA in 96 consecutive patients with a laboratory-confirmed diagnosis of COVID-19. Higher PTX3 plasma levels were observed in 52 (54.2%) patients admitted in ICU (median 21.0ng/mL, IQT 15.5-46.3 vs 12.4ng/mL IQT 6.1-20.2 in ward patients; p=0.0017) and in 22 (23%) patients died by 28 days (39.8ng/mL, IQT 20.2-75.7 vs 15.7ng/mL, IQT 8.2-21.6 in survivors; p=0.0001). After determining an optimal PTX3 cut-off for the primary outcome, the Kaplan-Meier curve showed an increased mortality in patients with PTX3>22.25ng/mL (Log-rank tests p<0.0001). In Cox regression model, PTX3>22.25ng/mL showed an adjusted Hazard Ratio (aHR) of 7.6 (95%CI2.45-23.76) in predicting mortality. Performing a multivariate logistic regression including all inflammatory markers (PTX3, ferritin, D-Dimer, IL-6, and CRP), PTX3 was the only marker significantly associated with death (aHR 1.13;95%CI1.02-1.24; p=0.021). The results reported here suggest that circulating and lung myelomonocytic cells are a major source of PTX3 and that PTX3 plasma levels can serve as a strong prognostic indicator of short-term mortality in COVID-19.

IgG serology in health care and administrative staff populations from 7 hospital representative of different exposures to SARS-CoV-2 in Lombardy, Italy (preprint)

Lombardy is one of the regions in Italy most affected by COVID-19. We assessed the diffusion of the virus via testing plasma anti-SARS-CoV-2 IgG antibodies in 3985 employees of 7 different hospitals, located across the Lombardy region in areas with different exposure to the epidemic. Subjects filled an anamnestic questionnaire to self-report on COVID-19 symptoms, co-morbidities, smoking, regular or smart-working, and the exposure to COVID-19-infected individuals. We show that the number of individuals exposed to the virus depended on the geographical area where the hospital was located and ranged between 3 to 43% which correlated with the incidence of COVID-19 in Lombardy. There was a higher prevalence of females than males positive for IgG, however the level of antibodies was similar, suggesting a comparable magnitude of the response. We observed 10% of IgG positive asymptomatic individuals and another 20% with one or two symptoms. 81% of individuals presenting both anosmia/ageusia and fever resulted SARS-CoV-2 infected. IgG positivity correlated with family contacts. In conclusion, the frequency of IgG positivity and SARS-CoV-2 infection is dependent on the geographical exposure to the virus and to extra-hospital exposure.

ACE2 and TMPRSS2 variants and expression as candidates to sex and country differences in COVID-19 severity in Italy (preprint)

Background: As the outbreak of coronavirus disease 2019 (COVID-19) progresses, prognostic markers for early identification of high-risk individuals are an urgent medical need. Italy has the highest rate of SARS-CoV-2 infection, the highest number of deaths, and the highest mortality rate among large countries. Worldwide, a more severe course of COVID-19 is associated with older age, comorbidities, and male sex. Hence, we searched for possible genetic components of the peculiar severity of COVID-19 among Italians, by looking at expression levels and variants in ACE2 and TMPRSS2 genes, which are crucial for viral infection. Methods: Exome and SNP array data from a large Italian cohort representative of the country's population were used to compare the burden of rare variants and the frequency of polymorphisms with European and East Asian populations. Moreover, we looked into gene expression databases to check for sex-unbalanced expression. Results: While we found no significant evidence that ACE2 is associated with disease severity/sex bias in the Italian population, TMPRSS2 levels and genetic variants proved to be possible candidate disease modulators, contributing to the observed epidemiological data among Italian patients. Conclusions: Our analysis suggests a role for TMPRSS2 variants and expression levels in modulating COVID-19 severity, a hypothesis that fosters a rapid experimental validation on large cohorts of patients with different clinical manifestations.