Biology subjects

Baldanti, F.

Publications and source records attributed to Baldanti, F..

At least 19 recordsLinked to original sources

bioRxiv2025

Fast and efficient Borrelia genome recovery from tick samples using Whole-Genome Amplification

Borrelia burgdorferi sensu lato bacteria are the causative agents of Lyme borreliosis, a multisystemic illness with an expanding epidemiology in temperate areas. Genomic studies on Borrelia are hindered by the difficulty of culturing procedures: standard protocols require extended incubation, substantial technical expertise, and are prone to failure, limiting timely recovery of isolates. This contributes to a low number of complete Borrelia genomes available in public repositories, particularly for species other than B. burgdorferi sensu stricto. Here we introduce a novel approach that overcomes the necessity for extended culture by utilizing Whole Genome Amplification (WGA) directly on freshly collected ticks, and that can be performed in parallel to classical culturing. The protocol is paired with a tailored bioinformatic pipeline designed to ensure accurate assembly and reliable downstream analyses. Benchmarking on multiple control isolates demonstrated that the method yields high-quality chromosomal assemblies. To demonstrate practical applicability, we applied the protocol to freshly collected ticks, successfully generating five high-quality Borrelia chromosomes (two B. lusitaniae, two B. afzelii and one B. garinii). By generating sequencing-ready DNA in five days rather than months, our protocol greatly streamlines the process and minimizes the effort associated with traditional culture-based methods. This workflow will facilitate broader representation of understudied Borrelia species and support future epidemiological, ecological, and evolutionary investigations on this pathogen.

genomics↗

bioRxiv2024

Genomic characterization of a persistent, azole-resistant C. parapsilosis strain responsible for a hospital outbreak during the first COVID-19 wave

Yeasts belonging to the Candida genus typically reside on the mucosal surface and within the respiratory and gastrointestinal tract as commensals. Under conditions of host vulnerability, they can act as opportunistic pathogens, leading to various forms of candidiasis, including candidemia. Such infections can be particularly problematic when caused by isolates that exhibit resistance to antifungal drugs, which is becoming more prevalent in many regions. One hundred and seven samples of Candida spp. were isolated from patients with candidemia in the hospital San Matteo in Pavia (Italy) over a period of 6 years, from 2015 to the first COVID wave in spring 2020. In order to understand the epidemiology of Candida infections in this hospital setting, the isolates were whole-genome sequenced which identified most as C. parapsilosis and C. albicans. Comparative genomics revealed that isolates of C. albicans were genomically diverse, indicating repeated introductions in the hospital from the community. C. parapsilosis isolates comprised two groups of highly similar isolates representing strains capable of long-term persistence in the hospital. All isolates of the main persistent group were resistant to fluconazole and presented variable levels of resistance to voriconazole and itraconazole, resulting from the Y132F substitution in erg11 and the N455D substitution in upc2. Interestingly, with the exception of the single isolate susceptible to both voriconazole and itraconazole, all the 61 isolates presented one unreported missense mutation in mrr1 (S1907C).

genomics↗

bioRxiv2024

Long-term Immunity of a Microneedle Array Patch of SARS-CoV-2 S1 Protein Subunit Vaccine Irradiated by Gamma Rays in Mice

COVID-19 vaccines effectively prevent symptomatic infection and severe disease, including hospitalization and death. However, unequal vaccine distribution during the pandemic, especially in low- and middle-income countries, has led to the emergence of vaccine-resistant strains. This underscores the need for alternative, safe, and thermostable vaccine platforms, such as dissolved microneedle array patches (MAP) delivering a subunit vaccine, which eliminate the need for cold chain and trained healthcare personnel. This study demonstrates that the SARS-CoV-2 S1 monomer with RS09, a TLR-4 agonist peptide, serves as an optimal protein subunit immunogen. This combination stimulates a stronger S1-specific immune response, resulting in binding to the membrane-bound spike on the cell surface and ACE2-binding inhibition, compared to the monomer S1 alone or trimer S1, regardless of RS09. MAP delivery of the rS1RS09 subunit vaccine elicited higher and longer-lasting immunity compared to conventional intramuscular injection. S1-specific IgG levels remained significantly elevated for up to 70 weeks post-administration. Additionally, different doses of 5, 15, and 45 g of MAP vaccines induced robust and sustained Th2-prevalent immune responses, suggesting a dose-sparing effect and inducing significantly high neutralizing antibodies against the Wuhan, Delta, and Omicron variants at 15 and 45 g dose. Moreover, gamma irradiation as a terminal sterilization method did not significantly affect immunogenicity, with irradiated vaccines maintaining comparable efficacy to non-irradiated ones. The stability of MAP vaccines was evaluated after long-term storage at room temperature and refrigeration for 19 months, showing minimal protein degradation. Further, after an additional one-month of storage at elevated temperature (42{degrees}C), rS1RS09 in both non-irradiated and irradiated MAP degraded less than 3%, while the liquid subunit vaccine degraded over 23%. Overall, these results indicate that gamma irradiation sterilized MAP-rS1RS09 vaccines maintain stability during extended storage without refrigeration, supporting their potential for mass production and widespread use in global vaccination efforts.

microbiology↗

bioRxiv2024

A newly developed whole genome sequencing protocol enables early tracking of Enterovirus D68 molecular evolution.

Background Human enterovirus D68 (EV-D68) has been associated with an increase in mild-to-severe pediatric respiratory diseases worldwide. The rate of circulation of this virus is largely underestimated in the population and genetic evolutionary data are usually available only for partial sequences (e.g VP1 region). To achieve a timely genomic surveillance, a reliable, high-throughput EV-68 sequencing assay is required. Here we report an improved high-throughput EV-D68 whole-genome sequencing (WGS) assay performed directly on clinical samples that is suitable for short-read sequencing platforms. Between June and December 2022, a total 37 (1.9%) respiratory samples were EV-D68 positive and together with 52 additional samples with a median cycle threshold of 28.3, ranging from 18 to 36.8 were included in the validation analyses. Overall, all the primers had good performance and no mismatches were detected in more than 85% of sequences (932 WGS dataset). Using a cut-off of Cq <32 in at least 85.5% of samples a WG or PG was obtained, confirming an acceptable positive sequencing rate for the designed method. A total of 65 WGS were obtained and have a mean coverage of 98.4% across the genome, with a median depth of 6158x (range 2815x-7560x). Based on the obtained data, this method is cost effective resulting in an easy-to-perform protocol helpful for tracing the evolution of EV-D68 in protein different from VP1. EV-D68 could become a significant pathogen for public health in the next future, and thus this protocol for whole genome sequencing could help clinical and molecular virologists to be ready for molecular epidemiology surveillance.

microbiology↗

bioRxiv2024

Second Boost of Omicron SARS-CoV-2 S1 Subunit Vaccine Induced Broad Humoral Immune Responses in Elderly Mice

Currently approved COVID-19 vaccines prevent symptomatic infection, hospitalization, and death from the disease. However, repeated homologous boosters, while considered a solution for severe forms of the disease caused by new SARS-CoV-2 variants in elderly individuals and immunocompromised patients, cannot provide complete protection against breakthrough infections. This highlights the need for alternative platforms for booster vaccines. In our previous study, we assessed the boost effect of the SARS-CoV-2 Beta S1 recombinant protein subunit vaccine (rS1Beta) in aged mice primed with an adenovirus-based vaccine expressing SARS-CoV-2-S1 (Ad5.S1) via subcutaneous injection or intranasal delivery, which induced robust humoral immune responses (1). In this follow-up study, we demonstrated that a second booster dose of a non-adjuvanted recombinant Omicron (BA.1) S1 subunit vaccine with Toll-like receptor 4 (TLR4) agonist RS09 (rS1RS09OM) was effective in stimulating strong S1-specific immune responses and inducing significantly high neutralizing antibodies against the Wuhan, Delta, and Omicron variants in 100-week-old mice. Importantly, the second booster dose elicits cross-reactive antibody responses, resulting in ACE2 binding inhibition against the spike protein of SARS-CoV-2 variants, including Omicron (BA.1) and its subvariants. Interestingly, the levels of IgG and neutralizing antibodies correlated with the level of ACE2 inhibition in the booster serum samples, although Omicron S1-specific IgG level showed a weaker correlation compared to Wuhan S1-specific IgG level. Furthermore, we compared the immunogenic properties of the rS1 subunit vaccine in young, middle-aged, and elderly mice, resulting in reduced immunogenicity with age, especially an impaired Th1-biased immune response in aged mice. Our findings demonstrate that the new variant of concern (VOC) rS1 subunit vaccine as a second booster has the potential to offer cross-neutralization against a broad range of variants and to improve vaccine effectiveness against newly emerging breakthrough SARS-CoV-2 variants in elderly individuals who were previously primed with the authorized vaccines.

immunology↗

bioRxiv2024

How to measure bacterial genome plasticity? A novel time-integrated index helps gather insights on pathogens

Genome plasticity can be defined as the capacity of a bacterial population to swiftly gain or lose genes. The time factor plays a fundamental role for the evolutionary success of microbes, particularly when considering pathogens and their tendency to gain antimicrobial resistance factors under the pressure of the extensive use of antibiotics. Multiple metrics have been proposed to provide insights into the gene content repertoire, yet they overlook the temporal component, which has a critical role in determining the adaptation and survival of a bacterial strain. In this study, we introduce a novel index that incorporates the time dimension to assess the rate at which bacteria exchange genes, thus fitting the definition of plasticity. Opposite to available indexes, our method also takes into account the possibility of contiguous genes being transferred together in one single event. We applied our novel index to measure plasticity in three widely studied bacterial species: Klebsiella pneumoniae, Staphylococcus aureus, and Escherichia coli. Our results highlight distinctive plasticity patterns in specific sequence types and clusters, suggesting a possible correlation between heightened genome plasticity and globally recognized high-risk clones. Our approach holds promise as an index for predicting the emergence of strains of potential clinical concern, possibly allowing for timely and more effective interventions. Impact statementHow quickly bacterial populations can acquire new functions is the key to their evolutionary success. This speed, called genome plasticity, is particularly relevant for human pathogens, especially when considering the acquisition of antimicrobial resistance. Today, the availability of large numbers of genomes from public databases makes it possible to develop a way to measure plasticity. However, none is currently available, besides indexes of gene content variability, which do not take into account the rate at which such gene content changes. In this work, we developed a plasticity index, called Flux Of Gene Segments (FOGS), and we tested it on large datasets of bacterial pathogen genomes. Interestingly, the subpopulations of the selected species that showed higher FOGS correspond to globally emerging high-risk clones. Therefore, we suggest that our index might be used not only to detect but also to predict emerging strains of human health concern. Data summaryThe authors confirm that all supporting data, code and protocols have been provided within the article or through supplementary data files.

genomics↗

bioRxiv2023

Fourth dose of Microneedle Array Patch of SARS-CoV-2 S1 Protein Subunit Vaccine Elicits Robust Long-lasting Humoral Responses in mice

The COVID-19 pandemic has underscored the pressing need for safe and effective booster vaccines, particularly in considering the emergence of new SARS-CoV-2 variants and addressing vaccine distribution inequalities. Dissolving microneedle array patches (MAP) offer a promising delivery method, enhancing immunogenicity and improving accessibility through the skins immune potential. In this study, we evaluated a microneedle array patch-based S1 subunit protein COVID-19 vaccine candidate, which comprised a bivalent formulation targeting the Wuhan and Beta variant alongside a monovalent Delta variant spike proteins in a murine model. Notably, the second boost of homologous bivalent MAP-S1(WU+Beta) induced a 15.7-fold increase in IgG endpoint titer, while the third boost of heterologous MAP-S1RS09Delta yielded a more modest 1.6-fold increase. Importantly, this study demonstrated that the administration of four doses of the MAP vaccine induced robust and long-lasting immune responses, persisting for at least 80 weeks. These immune responses encompassed various IgG isotypes and remained statistically significant for one year. Furthermore, neutralizing antibodies against multiple SARS-CoV-2 variants were generated, with comparable responses observed against the Omicron variant. Overall, these findings emphasize the potential of MAP-based vaccines as a promising strategy to combat the evolving landscape of COVID-19 and to deliver a safe and effective booster vaccine worldwide.

immunology↗

bioRxiv2023

P-DOR, an easy-to-use pipeline to reconstruct outbreaks using pathogen genomics

Bacterial Healthcare Associated Infections (HAIs) are a major threat worldwide, which can be counteracted by establishing effective infection control measures, guided by constant surveillance and timely epidemiological investigations. Genomics is crucial in modern epidemiology but lacks standard methods and user-friendly software, accessible to users without a strong bioinformatics proficiency. To overcome these issues we developed P-DOR, a novel tool for rapid bacterial outbreak characterization. P-DOR accepts genome assemblies as input, it automatically selects a background of publicly available genomes using k-mer distances and adds it to the analysis dataset before inferring a SNP-based phylogeny. Epidemiological clusters are identified considering the phylogenetic tree topology and SNP distances. By analyzing the SNP-distance distribution, the user can gauge the correct threshold. Patient metadata can be inputted as well, to provide a spatio-temporal representation of the outbreak. The entire pipeline is fast and scalable and can be also run on low-end computers. Availability and implementationP-DOR is implemented in Python3 and R and can be installed using conda environments. It is available from GitHub https://github.com/SteMIDIfactory/P-DOR under the GPL-3.0 license.

microbiology↗

bioRxiv2023

Conversion of monoclonal IgG to dimeric and secretory IgA restores neutralizing ability and prevents infection of Omicron lineages

The emergence of Omicron lineages and descendent subvariants continues to present a severe threat to the effectiveness of vaccines and therapeutic antibodies. We have previously suggested that an insufficient mucosal IgA response induced by the mRNA vaccines is associated with a surge in breakthrough infections. Here, we further show that the intramuscular mRNA and/or inactivated vaccines cannot sufficiently boost the mucosal sIgA response in uninfected individuals, particularly against the Omicron variant. We thus engineered and characterized recombinant monomeric, dimeric and secretory IgA1 antibodies derived from four neutralizing IgG monoclonal antibodies targeting the receptor-binding domain of the spike protein (01A05, rmAb23, DXP-604 and XG014). Compared to their parental IgG antibodies, dimeric and secretory IgA1 antibodies showed a higher neutralizing activity against different variants of concern (VOCs), in part due to an increased avidity. Importantly, the dimeric or secretory IgA1 form of the DXP-604 antibody significantly outperformed its parental IgG antibody, and neutralized the Omicron lineages BA.1, BA.2 and BA.4/5 with a 50-150-fold increase in potency, reaching the level of the most potent monoclonal antibodies described till date. In hACE2 transgenic mice, a single intranasal dose of the dimeric IgA DXP-604 conferred prophylactic and therapeutic protection against Omicron BA.5. Conversion of IgA and dimerization further enhanced or restored the neutralizing ability against the emerging Omicron sub-variants (DXP-604 for BQ.1, BQ.1.1 and BA2.75; 01A05 for BA2.75, BA.2.75.2 and XBB.1). Thus, dimeric or secretory IgA delivered by nasal administration may potentially be exploited for the treatment and prevention of Omicron infection, thereby providing an alternative tool for combating immune evasion by subvariants and, potentially, future VOCs. One Sentence SummaryEngineered dimeric and secretory IgA1 neutralized Omicron variant with higher potency than parental IgG.

microbiology↗

bioRxiv2023

Tetravalent SARS-CoV-2 S1 Subunit Protein Vaccination Elicits Robust Humoral and Cellular Immune Responses in SIV-Infected Rhesus Macaque Controllers

The COVID-19 pandemic has highlighted the need for safe and effective vaccines to be rapidly developed and distributed worldwide, especially considering the emergence of new SARS-CoV-2 variants. Protein subunit vaccines have emerged as a promising approach due to their proven safety record and ability to elicit robust immune responses. In this study, we evaluated the immunogenicity and efficacy of an adjuvanted tetravalent S1 subunit protein COVID-19 vaccine candidate composed of the Wuhan, B.1.1.7 variant, B.1.351 variant, and P.1 variant spike proteins in a nonhuman primate model with controlled SIVsab infection. The vaccine candidate induced both humoral and cellular immune responses, with T- and B cell responses mainly peaking post-boost immunization. The vaccine also elicited neutralizing and cross-reactive antibodies, ACE2 blocking antibodies, and T-cell responses, including spike specific CD4+ T cells. Importantly, the vaccine candidate was able to generate Omicron variant spike binding and ACE2 blocking antibodies without specifically vaccinating with Omicron, suggesting potential broad protection against emerging variants. The tetravalent composition of the vaccine candidate has significant implications for COVID-19 vaccine development and implementation, providing broad antibody responses against numerous SARS-CoV-2 variants.

immunology↗

bioRxiv2022

Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2 site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants. One sentence summaryBroadly cross-reactive antibodies that protect from SARS-CoV-2 variants are revealed by virus coldspot-driven discovery.

immunology↗

bioRxiv2022

SARS-CoV-2 S1 Subunit Booster Vaccination Elicits Robust Humoral Immune Responses in Aged Mice

Currently approved COVID-19 vaccines prevent symptomatic infection, hospitalization, and death of the disease. However, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants raises concerns of reduced vaccine effectiveness and increased risk of infection. Repeated homologous booster in elderly individuals and immunocompromised patients is considered to solve severe form of disease caused by new SARS-CoV-2 variants but cannot protect completely against breakthrough infection. In our previous study we assessed the immunogenicity of an adenovirus-based vaccine expressing SARS-CoV-2-S1 (Ad5.S1) in mice, resulting in that a single immunization with Ad5.S1, via subcutaneously injection or intranasal delivery, induced robust humoral and cellular immune responses [1]. As a follow up study, here we showed that vaccinated mice had high titers of anti-S1 antibodies at one year after vaccination compared to PBS immunized mice. Furthermore, one booster dose of non-adjuvanted recombinant S1Beta (rS1Beta) subunit vaccine was effective in stimulating strong long-lived S1-specific immune responses and inducing significantly high neutralizing antibodies against the Wuhan, Beta, and Delta strain with 3.6- to 19.5-fold change increases. Importantly, the booster dose elicits cross-reactive antibody responses resulting in ACE2 binding inhibition against spike of SARS-CoV-2 variants (Wuhan, Alpha, Beta, Gamma, Delta, Zeta, Kappa, New York, India) as early as two-week post-boost injection, persisting over 28 weeks after a booster vaccination. Interestingly, levels of neutralizing antibodies were correlated with not only level of S1-binding IgG but also level of ACE2 inhibition in the before- and after-booster serum samples. Our findings show that S1 recombinant protein subunit vaccine candidate as a booster has potential to offer cross-neutralization against broad variants, and has important implications for vaccine control of new emerging breakthrough SARS-CoV-2 variants in elderly individuals primed with adenovirus-based vaccine like AZD1222 and Ad26.COV2.S.

microbiology↗

bioRxiv2022

Adenovirus-Vectored SARS-CoV-2 Vaccine Expressing S1-N Fusion Protein

Additional COVID-19 vaccines that are safe, easy to manufacture, and immunogenic are needed for global vaccine equity. Here, we developed a recombinant type 5 adenovirus vector encoding for the SARS-CoV-2-S1 subunit antigen and nucleocapsid as a fusion protein (Ad5.SARS-CoV-2-S1N) delivered to BALB/c mice through multiple vaccine administration routes. A single subcutaneous (S.C.) immunization with Ad5.SARS-CoV-2-S1N induced a similar humoral response, along with a significantly higher S1-specific cellular response, as a recombinant type 5 adenovirus vector encoding for S1 alone (Ad5.SARS-CoV-2-S1). Immunogenicity was improved by homologous prime boost strategies, using either S.C. or intranasal (I.N.) delivery of Ad5.SARS-CoV-2-S1N, and further improved through heterologous prime boost, with traditional intramuscular (I.M.) injection, using subunit recombinant S1 protein. Priming with low dose (1x1010 v.p.) of Ad5.SARS-CoV-2-S1N and boosting with either wildtype recombinant rS1 or B.1.351 recombinant rS1 induced a robust neutralizing response, that was sustained against immune evasive Beta and Gamma SARS-CoV-2 variants, along with a long-lived plasma cell response in the bone marrow 29 weeks post vaccination. This novel Ad5-vectored SARS-CoV-2 vaccine candidate showed promising immunogenicity in mice and supports the further development of COVID-19 based vaccines incorporating the nucleoprotein as a target antigen.

immunology↗

bioRxiv2021

Immunity to SARS-CoV-2 up to 15 months after infection

BackgroundInformation concerning the longevity of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) following natural infection may have considerable implications for durability of immunity induced by vaccines. Here, we monitored the SARS-CoV-2 specific immune response in convalescent coronavirus disease-2019 (COVID-19) patients up to 15 months after symptoms onset. MethodsThe levels of anti-spike and anti-receptor binding domain antibodies and neutralizing activities were tested in a total of 188 samples from 136 convalescent patients who experience mild to critical COVID-19. Specific memory B and T cell responses were measured in 76 peripheral blood mononuclear cell samples collected from 54 patients. Twenty-three vaccinated individuals were included for comparison. FindingsFollowing a peak at day 15-28 post-infection, the IgG antibody response and plasma neutralizing titers gradually decreased over time but stabilized after 6 months. Plasma neutralizing activity against G614 was still detected in 87% of the patients at 6-15 months. Compared to G614, the median neutralizing titers against Beta, Gamma and Delta variants in plasma collected at early (15-103 days) and late (9-15 month) convalescence were 16- and 8-fold lower, respectively. SARS-CoV-2-specific memory B and T cells reached a peak at 3-6 months and persisted in the majority of patients up to 15 months although a significant decrease in specific T cells was observed between 6 and 15 months. ConclusionThe data suggest that antiviral specific immunity especially memory B cells in COVID-19 convalescent patients is long-lasting, but some variants of concern, including the fast-spreading Delta variant, may at least partially escape the neutralizing activity of plasma antibodies. FundingEU-ATAC consortium, the Italian Ministry of Health, the Swedish Research Council, SciLifeLab, and KAW.

immunology↗

bioRxiv2021

Bispecific antibody prevents SARS-CoV-2 escape and protects mice from disease

Neutralizing antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) are among the most promising approaches against coronavirus disease 2019 (COVID-19)1,2. We developed a bispecific, IgG1-like molecule (CoV-X2) based on two antibodies derived from COVID-19 convalescent donors, C121 and C1353. CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable S binding to Angiotensin-Converting Enzyme 2 (ACE2), the virus cellular receptor. Furthermore, CoV-X2 neutralizes SARS-CoV-2 and its variants of concern, as well as the escape mutants generated by the parental monoclonals. In a novel animal model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, combining into a single molecule the advantages of antibody cocktails.

immunology↗

bioRxiv2020

Neutrophil extracellular traps induce the epithelial-mesenchymal transition: implications in post-COVID-19 fibrosis

The release of neutrophil extracellular traps (NETs), a process termed NETosis, avoids pathogen spread but may cause tissue injury. NETs have been found in severe COVID-19 patients, but their role in disease development is still unknown. The aim of this study is to assess the capacity of NETs to drive epithelial-mesenchymal transition (EMT) of lung epithelial cells and to analyze the involvement of NETs in COVID-19. Neutrophils activated with PMA (PMA-Neu), a stimulus known to induce NETs formation, induce both EMT and cell death in the lung epithelial cell line, A549. Notably, NETs isolated from PMA-Neu induce EMT without cell damage. Bronchoalveolar lavage fluid of severe COVID-19 patients showed high concentration of NETs. Thus, we tested in an in vitro alveolar model the hypothesis that virus-induced NET may drive EMT. Co-culturing A549 at air-liquid interface with alveolar macrophages, neutrophils and SARS-CoV2, we demonstrated a significant induction of the EMT in A549 together with high concentration of NETs, IL8 and IL1{beta}, best-known inducers of NETosis. Lung tissues of COVID-19 deceased patients showed that epithelial cells are characterized by increased mesenchymal markers. These results show for the first time that NETosis plays a major role in triggering lung fibrosis in COVID-19 patients.

immunology↗

bioRxiv2020

Persistence of SARS-CoV-2 specific B- and T-cell responses in convalescent COVID-19 patients 6-8 months after the infection

BackgroundThe longevity of the immune response against SARS-CoV-2 is currently debated. We thus profiled the serum anti-SARS-CoV-2 antibody levels and virus specific memory B- and T-cell responses over time in convalescent COVID-19 patients. MethodsA cohort of COVID-19 patients from the Lombardy region in Italy who experienced mild to critical disease and Swedish volunteers with mild symptoms, were tested for the presence of elevated anti-spike and anti-receptor binding domain antibody levels over a period of eight months. In addition, specific memory B- and T-cell responses were tested in selected patient samples. ResultsAnti-SARS-CoV-2 antibodies were present in 85% samples collected within 4 weeks after onset of symptoms in COVID-19 patients. Levels of specific IgM or IgA antibodies declined after 1 month while levels of specific IgG antibodies remained stable up to 6 months after diagnosis. Anti-SARS-CoV-2 IgG antibodies were still present, though at a significantly lower level, in 80% samples collected at 6-8 months after symptom onset. SARS-CoV-2-specific memory B- and T-cell responses were developed in vast majority of the patients tested, regardless of disease severity, and remained detectable up to 6-8 months after infection. ConclusionsAlthough the serum levels of anti-SARS-CoV-2 IgG antibodies started to decline, virus-specific T and/or memory B cell responses increased with time and maintained during the study period (6-8 months after infection). FundingEuropean Unions Horizon 2020 research and innovation programme (ATAC), the Italian Ministry of Health, CIMED, the Swedish Research Council and the China Scholarship Council.

immunology↗

bioRxiv2020

Performance assessment of Respiratory Viral ELITe MGB(R) assay for the quantitative detection of influenza A/B and respiratory syncytial viruses

Influenza (Flu) and respiratory syncytial virus (RSV) are responsible for lower respiratory tract infections (LRTIs) associated with significant hospitalization among young children. In the present study, the performances of a triplex PCR assay detecting Flu A/B and RSV were compared with our in-house single-plex assays using 160 stored respiratory specimens previously tested using a panel of laboratory-developed real-time RT-PCR. Of them, 61 were positive for FluA, 41 for FluB, and 58 for RSV. All samples were retrospectively quantified with Respiratory Viral (RV) ELITe MGB(R) Panel (ELITechGroup Molecular Diagnostics, Puteaux, France) processed using ELITe InGenius(R) system. Overall, the total percentage agreement observed was 93.4% (57/61) for FluA, 92.7% (38/41) for FluB, and 86.2% (50/58) for RSV. A significant correlation of VL values was observed between the two methods for FluA and RSV ({rho}= 0.91 and 0.84). This finding was supported by the strength of agreement between the two methods, as showed by the linear regression analysis (R2 =0.84 and 0.80). FluB viral load values measured by RV Panel were less significantly correlated ({rho}= 0.77 and R2 =0.56). The bland-Altman analysis showed how 84.2% (48/57) of FluA and 86.0% of RSV (43/50) samples fell within {+/-}1.0 Log10 variation from our laboratory results, while only 21.1% (8/38) of FluB results fell within this range. The great majority of FluB samples (29/30) outside range had values higher than +1.0 Log10 (median +2.1 Log10 range +1.0 to +3.5 Log10). In conclusion, RV ELITe MGB(R) Panel constitutes a valid and robust system for simultaneous detection and quantification of Flu A/B and RSV.

microbiology↗