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Background: Mpox virus (MPXV) has recently spread outside of sub-Saharan Africa. This large multicentre study was conducted in Lombardy, the most densely populated Italian region accounting for more than 40% of Italian cases. The present study aims to: i) evaluate the presence and the shedding duration of MPXV DNA in different body compartments correlating the MPXV viability with the time to onset of symptoms; ii) provide evidence of MPXV persistence in different body compartment as a source of infection and iii) characterize the MPXV evolution by whole genome sequencing (WGS) during the outbreak occurred in Italy. Material and methods: The study included 353 patients with a laboratory-confirmed diagnosis of MPXV infection screened in several clinical specimens in the period May 24th - September 1st, 2022. Viral isolation was attempted from different biological matrices and complete genome sequencing was performed for 61 MPXV strains. Results: MPXV DNA detection was more frequent in the skin (94.4%) with the longest median time of viral clearance (16 days). The actively-replicating virus in cell culture was obtained for 123/377 (32.6%) samples with a significant higher viral quantity on isolation positive samples (20 vs 31, p

Introduction: Although the potential role of inanimate surfaces in SARS-CoV-2 transmission has yet to be adequately assessed, it is still routine practice to apply deep and expensive environmental disinfection protocols. The aim of this study was to verify the presence of viable virus on different surfaces exposed to droplets released by coughing in SARS-CoV-2 RNA positive patients. Methods: Patients admitted to hospital with a positive SARS-CoV-2 real-time (RT)-PCR swab were asked to cough on steel, cardboard, plastic and their hands. Surfaces were tested at baseline (T0) and at different timepoints thereafter using swabs dipped in medium, and quickly seeded on VERO E6 cells that were checked every other day for cytopathic effect (CPE). Laboratory-propagated SARS-CoV-2 strains were examined at the same time points and on identical materials. Results: Ten RNA-positive patients were enrolled into the study. The median cycle threshold value was 20.7 (range 13–28.3). Nasopharyngeal swabs from 3 of the patients yielded viable virus 2–10 days post-inoculation. However, in none of the patients was it possible to isolate viable SARS-CoV-2 from sputum under identical experimental conditions. A CPE was instead already visible using laboratory-propagated SARS-CoV-2 strains at 20′, 60′, 180′ while an effect at 24 h required a 6-day incubation. Conclusion: The evidence emerging from this real-life study suggests that droplets delivered by SARS-CoV-2 infected patients on common inanimate surfaces did not contain viable virus. In contrast, and in line with several laboratory-based experiments, in vitro adapted viruses could survive and grow on the same fomites.

ABSTRACT The coronavirus disease 2019 (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 severe acute respiratory syndrome coronavirus 2 (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, angiotensin-converting enzyme 2 (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. IMPORTANCE The study provides important insights into the immunogenicity and efficacy of a tetravalent protein subunit vaccine candidate against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The vaccine induced both humoral and cellular immune responses in nonhuman primates with controlled SIVagm infection and was able to generate Omicron variant-specific antibodies without specifically vaccinating with Omicron. These findings suggest that the tetravalent composition of the vaccine candidate could provide broad protection against multiple SARS-CoV-2 variants while minimizing the risk of immune escape and the emergence of new variants. Additionally, the use of rhesus macaques with controlled SIVsab infection may better represent vaccine immunogenicity in humans with chronic viral diseases, highlighting the importance of preclinical animal models in vaccine development. Overall, the study provides valuable information for the development and implementation of coronavirus disease 2019 vaccines, particularly for achieving global vaccine equity and addressing emerging variants.

ABSTRACT The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has raised concerns about reduced vaccine effectiveness and the increased risk of infection, and while repeated homologous booster shots are recommended for elderly and immunocompromised individuals, they cannot completely protect against breakthrough infections. In our previous study, we assessed the immunogenicity of an adenovirus-based vaccine expressing SARS-CoV-2 S1 (Ad5.S1) in mice, which induced robust humoral and cellular immune responses (E. Kim, F. J. Weisel, S. C. Balmert, M. S. Khan, et al., Eur J Immunol 51:1774–1784, 2021, https://doi.org/10.1002/eji.202149167). In this follow-up study, we found that the mice had high titers of anti-S1 antibodies 1 year after vaccination, and one booster dose of the nonadjuvanted rS1Beta (recombinant S1 protein of SARS-CoV-2 Beta [B.1.351]) subunit vaccine was effective at stimulating strong long-lived S1-specific immune responses and inducing significantly high neutralizing antibodies against Wuhan, Beta, and Delta strains, with 3.6- to 19.5-fold increases. Importantly, the booster dose also elicited cross-reactive antibodies, resulting in angiotensin-converting enzyme 2 (ACE2) binding inhibition against spikes of SARS-CoV-2, including Omicron variants, persisting for >28 weeks after booster vaccination. Interestingly, the levels of neutralizing antibodies were correlated not only with the level of S1 binding IgG but also with ACE2 inhibition. Our findings suggest that the rS1Beta subunit vaccine candidate as a booster has the potential to offer cross-neutralization against broad variants and has important implications for the vaccine control of newly emerging breakthrough SARS-CoV-2 variants in elderly individuals primed with adenovirus-based vaccines like AZD1222 and Ad26.COV2.S. IMPORTANCE Vaccines have significantly reduced the incidences of severe coronavirus disease 2019 (COVID-19) cases and deaths. However, the emergence of SARS-CoV-2 variants has raised concerns about their increased transmissibility and ability to evade neutralizing antibodies, especially among elderly individuals who are at higher risks of mortality and reductions of vaccine effectiveness. To address this, a heterologous booster vaccination strategy has been considered as a solution to protect the elderly population against breakthrough infections caused by emerging variants. This study evaluated the booster effect of an S1 subunit vaccine in aged mice that had been previously primed with adenoviral vaccines, providing valuable preclinical evidence for elderly people vaccinated with the currently approved COVID-19 vaccines. This study confirms the potential for using the S1 subunit vaccine as a booster to enhance cross-neutralizing antibodies against emerging variants of concern.

Several COVID-19 vaccines have shown good efficacy in clinical trials. Here, the authors provide real world effectiveness data in a group of BNT162b2 vaccinated health care workers and find that breakthrough infections are asymptomatic or mild.

Orofacial clefts (OFC) are frequent congenital malformations characterized by insufficient separation of oral and nasal cavities and require presurgical infant orthopedics and surgical interventions within the first year of life. Wound healing disorders and higher prevalence of gingivitis and plaque levels are well-known challenges in treatment of children with OFC. However, oral inflammatory mediators were not investigated after birth using non-invasive sampling methods so far. In order to investigate the impact of OFC on oral cytokine levels, we collected tongue smear samples from 15 neonates with OFC and 17 control neonates at two time points (T), T0 at first consultation after birth, and T1, 4 to 5 weeks later. The samples were analyzed using multiplex immunoassay. Overall, we found significantly increased cytokine levels (TNF, IL-1β/-2/-6/-8/-10) in tongue smear samples from neonates with OFC compared to controls, especially at T0. The increase was even more pronounced in neonates with a higher cleft severity. Further, we detected a significant positive correlation between cleft severity score and distinct pro-inflammatory mediators (GM-CSF, IL-1β, IL-6, IL-8) at T0. Further, we found that breast-milk (bottle) feeding was associated with lower levels of pro-inflammatory cytokines (IL-6/-8) in neonates with OFC compared to formula-fed neonates. Our study demonstrated that neonates with OFC, especially with high cleft severity, are characterized by markedly increased inflammatory mediators in tongue smear samples within the first weeks of life potentially presenting a risk for oral inflammatory diseases. Therefore, an inflammatory monitoring of neonates with (severe) OFC and the encouragement of mother to breast-milk (bottle) feed might be advisable after birth and/or prior to cleft surgery.

Objective: The protection from SARS-CoV-2 infection induced by SARS-CoV-2 anti-S1 and anti-S2 IgG antibody positivity resulting from natural infection was evaluated. Methods: The frequency of SARS-CoV-2 infection (as determined by virus RNA detection) was evaluated in a group of 1,460 seropositive and a control group of 8,150 seronegative healthcare workers in three Centres of Northern Italy in the period June-November 2020. Neutralizing serum titers were analyzed in seropositive subjects with or without secondary SARS-CoV-2 infection. Results: During the 6-month survey, 1.78% seropositive subjects developed secondary SARS-CoV-2 infection while 6.63% seronegative controls developed primary infection (odds ratio: 0.26; 95% confidence interval: 0.17-0.38). Secondary infection was associated with low or absent serum neutralizing titer (p

Abstract Background Convalescent plasma (CP) and hyperimmune plasma (HP) are passive immunotherapies consisting in the infusion of plasma from recovered people into infected patients. Following pre-existing evidence in many other viral diseases, such as SARS, MERS and Ebola, CP and HP have also been proposed for the treatment of COVID-19. Nevertheless, due to the lack of large, well-designed, clinical trials, no clear-cut guidelines exist about what subtype of patient CP and HP should be administered to. Case presentation We have reported the cases of 3 patients, all immunosuppressed and affected by non-severe, prolonged COVID-19. They were treated with HP, whose neutralizing titer was higher than 1/80. The first patient was a 55-year-old male, who had undergone lung transplant. He was under therapy with Tacrolimus and developed non-neutralizing antibodies against SARS-CoV2. The second patient was a 77-year-old female, affected by follicular lymphoma. She had tested positive for SARS-CoV2 after 6 months. The third was a 60-year-old patient, affected by chronic leukemia. He did not develop antibodies after 2-month disease. All 3 patients received HP and had tested negative for SARS-CoV2 within 2 weeks. Conclusion Despite encouraging initial data, no strong evidence exist in support of CP and HP to treat COVID-19. In our experience, although limited due to the reduced number of patients, we found a good safety and efficacy of HP in 3 immuno-deficient subjects. Further data are needed in order to assess whether this subtype of patients may particularly benefit from passive immunization.

The role and durability of the immunogenicity of the BNT162b2 mRNA vaccine against severe acute respiratory virus 2 (SARS-CoV-2), in cancer patients one year after receiving the third dose have to be elucidated. We have prospectively evaluated the long-term immunogenicity of the third dose of the SARS-CoV-2 BNT162b2 mRNA vaccine in 55 patients undergoing active treatment. Neutralizing antibody (NT Ab) titers against Omicron variants and total anti-trimeric S IgG levels were measured one year after the third dose. Heparinized whole-blood samples were used for the assessment of the SARS-CoV-2 interferon-γ release assay (IGRA). Thirty-seven patients (67.3%) showed positive total anti-trimeric S IgG one year after the third dose. Looking at the T-cell response against the spike protein, the frequency of responder patients did not decrease significantly between six and twelve months after the third dose. Finally, less than 20% of cancer patients showed an undetectable NT Ab titer against BA.1 and BA.5 variants of concern (VOCs). Underlying therapies seem to not affect the magnitude or frequency of the immune response. Our work underlines the persistence of humoral and cellular immune responses against BNT162b2 in a cohort of cancer patients one year after receiving the third dose, regardless of the type of underlying therapy.

Abstract Anti‐inflammatory and immune‐modulatory therapies have been proposed for the treatment of COVID‐19 and its most serious complications. Among others, the use of mesenchymal stromal cells (MSCs) is under investigation given their well‐documented anti‐inflammatory and immunomodulatory properties. However, some critical issues regarding the possibility that MSCs could be infected by the virus have been raised. Angiotensin‐converting enzyme 2 (ACE2) and type II transmembrane serine protease (TMPRSS2) are the main host cell factors for the severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2), entry, but so far it is unclear if human MSCs do or do not express these two proteins. To elucidate these important aspects, we evaluated if human MSCs from both fetal and adult tissues constitutively express ACE2 and TMPRSS2 and, most importantly, if they can be infected by SARS‐CoV‐2. We evaluated human MSCs derived from amnios, cord blood, cord tissue, adipose tissue, and bone marrow. ACE2 and TMPRSS2 were expressed by the SARS‐CoV‐2‐permissive human pulmonary Calu‐3 cell line but not by all the MSCs tested. MSCs were then exposed to SARS‐CoV‐2 wild strain without evidence of cytopathic effect. Moreover, we also excluded that the MSCs could be infected without showing lytic effects since their conditioned medium after SARS‐CoV‐2 exposure did not contain viral particles. Our data, demonstrating that MSCs derived from different human tissues are not permissive to SARS‐CoV‐2 infection, support the safety of MSCs as potential therapy for COVID‐19.

Real-time reverse transcription PCR is currently the most sensitive method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Defining whether a patient could be contagious or not contagious in the presence of residual SARS-CoV-2 RNA is of extreme importance in the context of public health. In this prospective multicenter study, virus isolation was prospectively attempted in 387 nasal swabs from clinically recovered patients showing low viral load (quantification cycle, Cq, value greater than 30). The median Cq value was 36.8 (range 30.0–39.4). Overall, a cytopathic effect was detected in nine samples, corresponding to a culture positivity rate of 2.3% (9/387). The results of this study help to dissect true virus replication and residual viral RNA detection in recovered patients.

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic proportions. Given that the main target of SARS-CoV-2 are lungs leading to severe pneumonia with hyperactivation of the inflammatory cascade, we conducted a prospective study to assess alveolar inflammatory status in patients with moderate to severe COVID-19. Methods Diagnostic bronchoalveolar lavage (BAL) was performed in 33 adult patients with SARS-CoV-2 infection by real-time PCR on nasopharyngeal swab admitted to the Intensive care unit (ICU) (n = 28) and to the Intermediate Medicine Ward (IMW) (n = 5). We analyze the differential cell count, ultrastructure of cells and Interleukin (IL)6, 8 and 10 levels. Results ICU patients showed a marked increase in neutrophils (1.24 × 105 ml− 1, 0.85–2.07), lower lymphocyte (0.97 × 105 ml− 1, 0.024–0.34) and macrophages fractions (0.43 × 105 ml− 1, 0.34–1.62) compared to IMW patients (0.095 × 105 ml− 1, 0.05–0.73; 0.47 × 105 ml− 1, 0.28–1.01 and 2.14 × 105 ml− 1, 1.17–3.01, respectively) (p

Summary: Information concerning the longevity of immunity to 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 COVID-19 patients followed up to 15 months after symptoms onset. Following a peak at day 15–28 postinfection, the IgG antibody response and plasma neutralizing titers gradually decreased over time but stabilized after 6 months. Compared to G614, plasma neutralizing titers were more than 8-fold lower against variants Beta, Gamma, and Delta. SARS-CoV-2-specific memory B and T cells persisted in the majority of patients up to 15 months although a significant decrease in specific T cells, but not B cells, was observed between 6 and 15 months. Antiviral specific immunity, especially memory B cells in COVID-19 convalescent patients, is long-lasting, but some variants of concern may at least partially escape the neutralizing activity of plasma antibodies.

Abstract Background Unloading/disuse induces skeletal muscle atrophy in bedridden patients and aged people, who cannot prevent it by means of exercise. Because interventions against known atrophy initiators, such as oxidative stress and neuronal NO synthase (nNOS) redistribution, are only partially effective, we investigated the involvement of melusin, a muscle‐specific integrin‐associated protein and a recognized regulator of protein kinases and mechanotransduction in cardiomyocytes. Methods Muscle atrophy was induced in the rat soleus by tail suspension and in the human vastus lateralis by bed rest. Melusin expression was investigated at the protein and transcript level and after treatment of tail‐suspended rats with atrophy initiator inhibitors. Myofiber size, sarcolemmal nNOS activity, FoxO3 myonuclear localization, and myofiber carbonylation of the unloaded rat soleus were studied after in vivo melusin replacement by cDNA electroporation, and muscle force, myofiber size, and atrogene expression after adeno‐associated virus infection. In vivo interference of exogenous melusin with dominant‐negative kinases and other atrophy attenuators (Grp94 cDNA; 7‐nitroindazole) on size of unloaded rat myofibers was also explored. Results Unloading/disuse reduced muscle melusin protein levels to about 50%, already after 6 h in the tail‐suspended rat (P < 0.001), and to about 35% after 8 day bed rest in humans (P < 0.05). In the unloaded rat, melusin loss occurred despite of the maintenance of β1D integrin levels and was not abolished by treatments inhibiting mitochondrial oxidative stress, or nNOS activity and redistribution. Expression of exogenous melusin by cDNA transfection attenuated atrophy of 7 day unloaded rat myofibers (−31%), compared with controls (−48%, P = 0.001), without hampering the decrease in sarcolemmal nNOS activity and the increase in myonuclear FoxO3 and carbonylated myofibers. Infection with melusin‐expressing adeno‐associated virus ameliorated contractile properties of 7 day unloaded muscles (P ≤ 0.05) and relieved myofiber atrophy (−33%) by reducing Atrogin‐1 and MurF‐1 transcripts (P ≤ 0.002), despite of a two‐fold increase in FoxO3 protein levels (P = 0.03). Atrophy attenuation by exogenous melusin did not result from rescue of Akt, ERK, or focal adhesion kinase activity, because it persisted after co‐transfection with dominant‐negative kinase forms (P < 0.01). Conversely, melusin cDNA transfection, combined with 7‐nitroindazole treatment or with cDNA transfection of the nNOS‐interacting chaperone Grp94, abolished 7 day unloaded myofiber atrophy. Conclusions Disuse/unloading‐induced loss of melusin is an early event in muscle atrophy which occurs independently from mitochondrial oxidative stress, nNOS redistribution, and FoxO3 activation. Only preservation of melusin levels and sarcolemmal nNOS localization fully prevented muscle mass loss, demonstrating that both of them act as independent, but complementary, master switches of muscle disuse atrophy.

We reported the long-term kinetics of immune response after vaccination and evaluated the immunogenicity after a third dose of mRNA vaccine in 86 healthcare workers. Humoral response was analyzed by measuring anti-spike IgG and SARS-CoV-2 NTAbs titer; cell-mediated response was measured as frequency of IFN-γ producing T-cells and cell proliferation. Memory B cells secreting SARS-CoV-2 RBD-IgG were measured by B-spot assay. At three weeks after the third dose (T4), the frequency of subjects showing NT-Abs titer at the upper detection limit (≥640) was significantly higher than that observed at three weeks after the second dose (26/77; 33.7% vs. 9/77; 11.6%; p = 0.0018). Additionally, at T4, all the subjects reached positive levels of T-cell mediated response (median 110 SFU/106 PBMC, IQR 73-231). While the number of IFNγ-producing T-cells decreased between second and third dose administration, the T-cell proliferative response did not decrease but was sustained during the follow-up. Among T-cell subsets, a higher proliferative response was observed in CD4+ than in CD8+ population. Moreover, even if a decline in antibody response was observed between the second and third dose, a sustained persistence of memory B cells was observed. Subsequently, the third dose did not affect the frequency of memory B cells, while it restored or increased the peak antibody levels detected after the second dose.

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. Bronchoalveolar lavage fluid of severe COVID-19 patients showed high concentration of NETs that correlates with neutrophils count; moreover, the analysis of lung tissues of COVID-19 deceased patients showed a subset of alveolar reactive pneumocytes with a co-expression of epithelial marker and a mesenchymal marker, confirming the induction of EMT mechanism after severe SARS-CoV2 infection. By airway in vitro models, cultivating A549 or 16HBE at air-liquid interface, adding alveolar macrophages (AM), neutrophils and SARS-CoV2, we demonstrated that to trigger a complete EMT expression pattern are necessary the induction of NETosis by SARS-CoV2 and the secretion of AM factors (TGF-β, IL8 and IL1β). All our results highlight the possible mechanism that can induce lung fibrosis after SARS-CoV2 infection.

COVID-19 convalescent plasma (CCP) has been the only specific anti-viral therapy against SARS-CoV-2 available for more than one year. Following the negative results from most randomized controlled trials on its efficacy in COVID-19 hospitalized patients and the availability of anti-spike monoclonal antibodies (mAbs), the use of CCP has subsequently rapidly faded. However, the continuous appearance of new variants of concern (VOCs), most of which escape mAbs and vaccine-elicited neutralizing antibodies (nAbs), has renewed the interest towards CCP, at least in seronegative immunocompetent patients, and in immunocompromised patients not able to mount a protective immune response. We report here the experience of a single Italian hospital in collecting and transfusing CCP in immunocompromised patients hospitalized for severe COVID-19 between October 2021 and March 2022. During this 6-month period, we collected CCP from 32 vaccinated and convalescent regular blood donors, and infused high nAb-titer CCP units (titered against the specific VOC affecting the recipient) to 21 hospitalized patients with severe COVID-19, all of them seronegative at the time of CCP transfusion. Patients’ median age was 66 years (IQR 50–74 years) and approximately half of them (47.6%, 10/21) were immunocompromised. Two patients were rescued after previous failure of mAbs. No adverse reactions following CCP transfusion were recorded. A 28-day mortality rate of 14.3 percent (3/21) was reported, with age, advanced disease stage and late CCP transfusion associated with a worse outcome. This real-life experience also supports the use of CCP in seronegative hospitalized COVID-19 patients during the Delta and Omicron waves.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has severely impacted on public health, mainly on immunosuppressed patients, including solid organ transplant recipients. Vaccination represents a valuable tool for the prevention of severe SARS-CoV-2 infection, and the immunogenicity of mRNA vaccines has been evaluated in transplanted patients. In this study, we investigated the role of a third dose of the BNT162b2 vaccine in a cohort of kidney transplant recipients, analyzing both humoral and cell-mediated responses. We observed an increased immune response after the third dose of the vaccine, especially in terms of Spike-specific T cell response. The level of seroconversion remained lower than 50% even after the administration of the third dose. Mycophenolate treatment, steroid administration and age seemed to be associated with a poor immune response. In our cohort, 11/45 patients experienced a SARS-CoV-2 infection after the third vaccine dose. HLA antibodies appearance was recorded in 7 out 45 (15.5%) patients, but none of the patients developed acute renal rejection. Further studies for the evaluation of long-term immune responses are still ongoing, and the impact of a fourth dose of the vaccine will be evaluated.

SARS-CoV-2 still represents a global health burden, causing more than six million deaths worldwide. Moreover, the emergence of new variants has posed new issues in terms of vaccine efficacy and immunogenicity. In this study, we aimed to evaluate the neutralizing antibody response against SARS-CoV-2 variants in different cohorts of vaccinated and unvaccinated subjects. Four-fold diluted sera from SARS-CoV-2 naïve and recovered subjects vaccinated with two or three doses of the BNT162b2 vaccine were challenged against 14 SARS-CoV-2 variants, and the SARS-CoV-2 neutralizing antibody titer was measured. Results were compared with those obtained from unvaccinated COVID-19 recovered patients. Overall, a better SARS-CoV-2 NT Abs response was observed in recovered vaccinated subjects after three doses of the vaccine when compared to unvaccinated patients and vaccinated subjects with only two doses. Additionally, the lowest level of response was observed against the Omicron variant. In conclusion, third doses of BNT162b2 vaccine seems to elicit a sustained response against the large majority of variants.

Abstract Background West Nile virus (WNV) is a mosquito-borne RNA virus belonging to the Flaviviridae family. Symptomatic infection happens in only about 20% of the cases, while WNV neuroinvasive disease (WNND) is rare and accounts for less than 1%. There is insufficient information about natural history and clinical course in children, because underdiagnosis is common, and reports are scarce. On the other hand, Europe has seen a dramatic increase of WNV infections in the last decades, and the Po valley itself, in Northern Italy, has become an endemic region since 2013. Case presentation We hereby report a case of West-Nile virus neuroinvasive disease in a 12-year-old boy. This is one of the very few cases diagnosed in the Italian pediatric population. The clinical presentation was compatible with acute encephalitis. Diagnosis was made by detection of specific IgM in both serum and cerebrospinal fluid. He finally was discharged with complete recovery, and no neurologic sequelae after a 12-months follow up period. Conclusions Given its non-specific clinical presentation, the diffusion of WNV constitutes a crucial and emerging concern. Even though rare, neuroinvasive WNV infection should always be suspected in pediatric patients, living or traveling in endemic areas, presenting with meningitis, encephalitis or acute flaccid paralysis during the WNV transmission season.

Hyperimmune plasma from Covid-19 convalescent is a potential treatment for severe Covid-19. We conducted a multicenter one arm proof of concept interventional study. Patients with Covid-19 disease with moderate-to-severe Acute Respiratory Distress Syndrome, elevated C-reactive Protein and need for mechanical ventilation and/or CPAP were enrolled. One to three 250-300 ml unit of hyperimmune plasma (neutralizing antibodies titer ≥1:160) were administered. Primary outcome was 7-days hospital mortality. Secondary outcomes were PaO2/FiO2, laboratory and radiologic changes, as well as weaning from mechanical ventilation and safety. The study observed 46 patients from March, 25 to April, 21 2020. Patients were aged 63, 61% male, of them, 30 were on CPAP and 7 intubated. PaO2/FiO2 was 128 (SD 47). Bilateral infiltrates on chest X-ray was present in 36 patients (84%). Symptoms and ARDS duration were 14 (SD 7) and 6 days (SD 3). Three patients (6.5%) died within 7 days as compared to an expected 15% from the National Statistics and 30% from a small concurrent cohort of 23 patients. The upper one-sided 90%CI was 13.9%, allowing to reject the null hypothesis of a 15% mortality. PaO2/FiO2 increased by 112 units (95%CI 82 to142) in survivors, the chest radiogram severity decreased in 23% (95%CI 5% to 42%); CRP, Ferritin and LDH decreased by 60, 36 and 20% respectively. Weaning from CPAP was obtained in 26/30 patients and 3/7 were extubated. Five serious adverse events occurred in 4 patients (2 likely, 2 possible treatment related). In conclusion, Hyperimmune plasma in Covid-19 shows promising benefits, to be confirmed in a randomized controlled trial. This proof of concept study could open to future developments including hyperimmune plasma banking, development of standardized pharmaceutical products and monoclonal antibodies.

During the current pandemic of COVID-19 caused by the new Coronavirus SARS-CoV-2, the confinement measures slowed down the contagion, but did not completely avoid the disease diffusion for health workers, patients and the remaining population. The individual protection equipment (e.g. facial masks), filters for air conditioning systems and for medical respiratory devices do not possess an intrinsic antimicrobial/virucidal action and they are susceptible to microbial/viral colonization. An efficient antimicrobial/virucidal technology on air filtering media is crucial for maintaining a safe air environment and protecting people, in particular when lockdown is eased. This short communication reports about the virucidal effect, preliminary verified towards Coronavirus SARS-CoV-2, of a silver nanocluster/silica composite sputtered coating, directly applicated on a FFP3 mask.

BackgroundZika virus has recently spread to South- and Central America, causing congenital birth defects and neurological complications. Many people at risk are flavivirus pre-immune due to prior infections with other flaviviruses (e.g. dengue virus) or flavivirus vaccinations. Since pre-existing cross-reactive immunity can potentially modulate antibody responses to Zika virus infection and may affect the outcome of disease, we analyzed fine-specificity as well as virus-neutralizing and infection-enhancing activities of antibodies induced by a primary Zika virus infection in flavivirus-naïve as well as yellow fever- and/or tick-borne encephalitis-vaccinated individuals.MethodologyAntibodies in sera from convalescent Zika patients with and without vaccine-induced immunity were assessed by ELISA with respect to Zika virus-specificity and flavivirus cross-reactivity. Functional analyses included virus neutralization and infection-enhancement. The contribution of IgM and cross-reactive antibodies to these properties was determined by depletion experiments.Principal findingsPre-existing flavivirus immunity had a strong influence on the antibody response in primary Zika virus infections, resulting in higher titers of broadly flavivirus cross-reactive antibodies and slightly lower levels of Zika virus-specific IgM. Antibody-dependent enhancement (ADE) of Zika virus was mediated by sub-neutralizing concentrations of specific IgG but not by cross-reactive antibodies. This effect was potently counteracted by the presence of neutralizing IgM. Broadly cross-reactive antibodies were able to both neutralize and enhance infection of dengue virus but not Zika virus, indicating a different exposure of conserved sequence elements in the two viruses.ConclusionsOur data point to an important role of flavivirus-specific IgM during the transient early stages of infection, by contributing substantially to neutralization and by counteracting ADE. In addition, our results highlight structural differences between strains of Zika and dengue viruses that are used for analyzing infection-enhancement by cross-reactive antibodies. These findings underscore the possible impact of specific antibody patterns on flavivirus disease and vaccination efficacy.

The immunogenicity of severe acute respiratory syndrome 2 virus (SARS-CoV-2) vaccines in immunocompromised patients remains to be further explored. Here, we evaluated the immunogenicity elicited by complete vaccination with BNT162b2 vaccine in solid organ transplant recipients (SOTRs). A cohort of 110 SOTRs from Northern Italy were vaccinated with two doses of BNT162b2 mRNA vaccine and prospectively monitored at baseline and after 42 days. Both SARS-CoV-2 naïve and recovered subjects were included. Humoral response elicited by vaccination, including SARS-CoV-2 neutralizing antibodies (SARS-CoV-2 NT Abs), was evaluated; additionally, ex-vivo ELISpot assay was performed for the quantification of Spike-specific T-cell response. Results were compared with those obtained in a cohort of healthy subjects. In a subset of patients, humoral and T-cell responses against delta variant were also evaluated. Less than 20% of transplanted subjects developed a positive humoral and cell-mediated response after complete vaccination schedule. Overall, median levels of immune response elicited by vaccination were significantly lower with respect to controls in SARS-CoV-2 naïve transplant, but not in SARS-CoV-2 recovered transplanted patients. Additionally, a significant impairment of both humoral and cell-mediated response was observed in mycophenolate-treated patients. Positive delta-SARS-CoV-2 NT Abs levels were detected in almost all the SARS-CoV-2 recovered subjects but not in previously uninfected patients. Our study supports previous observations of a low level of seroconversion after vaccination in transplanted patients.

We aimed to explore whether variants of SARS-CoV-2 (Chinese-derived strain (D614, lineage A), Italian strain PV10734 (D614G, lineage B.1.1) and Alpha strain (lineage B.1.1.7)) were able to infect monocytes (MN) and monocyte-derived macrophages (MDM) and whether these infected cells may, in turn, be vectors of infection. For this purpose, we designed an in vitro study following the evolution of MN and MDM infection at different time points in order to confirm whether these cells were permissive for SARS-CoV-2 replication. Finally, we investigated whether, regardless of viral replication, the persistent virus can be transferred to non-infected cells permissive for viral replication. Thus, we co-cultured the infected MN/MDM with permissive VERO E6 cells verifying the viral transmission. This is a further in vitro demonstration of the important role of MN and MDM in the dissemination of SARS-CoV-2 and evolution of the COVID-19 disease.

COVID-19 related morbidity and mortality have been often attributed to an exaggerated immune response. The role of cytokines and chemokines in COVID-19 and their contributions to illness severity are known, and thus their profiling from patient bronchoalveolar lavage (BAL) samples would help in understanding the disease progression. To date, limited studies have been performed on COVID-19 BAL samples, as the manipulation of such specimens (potentially containing live viruses) requires several laboratorial precautions, such as personnel training and special equipment, a requirement that not all laboratories can fulfil. Here, we assessed two fast and easily applicable methods (ultrafiltration and ultraviolet–C irradiation) for their impact on viral load removal or inactivation, respectively and on cytokine profiles preservation. Eight samples of BAL fluids from SARS-CoV2 patients with high viral load were tested. For both methods, complete removal was confirmed by lack of viral replication in Vero E6 cells and by RT-qPCR. Although both methods showed to remove completely the active SARS-CoV2 viral load, only UVC treatment has little or no quantitative effect on total cytokines/chemokines measurements, however cytokines profile and relative ratios are preserved or minimally altered when compared data obtained by the two different decontamination methods. Sample preparation and manipulation can greatly affect the analytical results; therefore, understanding if changes occurred after sample processing is of outmost importance for reliable data and can be useful to improve clinical practice.

Phleboviruses transmitted by phlebotomine sandflies are endemic in the Mediterranean basin. Toscana phlebovirus (TOSV), Sicilian phlebovirus (SFSV), and Naples phlebovirus (SFNV) are responsible of summer fever, with well-known pathogenic potential for humans ranging from asymptomatic to mild fever, in addition to neuro-invasive infections during summer. Although TOSV, in particular, is a significant and well-known human pathogen, SFVs remain neglected, with many gaps in the relevant knowledge. Sero-epidemiological studies and case reports recently showed a geographical wider distribution than previously considered, although the real incidence of phleboviruses infections in the Mediterranean area is still unknown. Here we retrospectively evaluated the circulation of phleboviruses during summer seasons between 2007 and 2019 in 649 patients showing neurological symptoms using both molecular and serological approaches. We found that 42/649 (6.5%) subjects experienced phlebovirus infection and only 10/42 cases were detected by molecular assays, whereas the other 32/42 were identified using serological approaches, including neutralization assays. During the 2013 summer, an outbreak in the Lombardy region is described because the prevalence of phlebovirus infection reached 37.2% (19/51 subjects). Interestingly, only 5/19 (26.5%) reported traveling in endemic areas. Of note, no cross-neutralization was observed between different strains tested, showing the possibility to be reinfected by newly discovered phlebovirus strains. In conclusion, phlebovirus infections are still inadequately considered by physicians and are generally underestimated. However, based on our results, sandfly fever viruses should be routinely included in diagnostic panels during summer period, including in Northern Italy.

West Nile virus (WNV) and Usutu virus (USUV) are two related arboviruses (genus Flavivirus, family Flaviviridae), with birds as a reservoir and mosquitoes as transmitting vectors. In recent years, WNV epidemiology changed in many European countries with increased frequency of outbreaks posing the issue of virus transmission risks by blood transfusion. USUV emerged for the first time in birds of the Tuscany region (Italy) in 1996 and in 2001 in Austria. While WNV is responsible for both mild and neuroinvasive diseases, USUV infection is usually asymptomatic and neuroinvasive symptoms are rare. Since WNV and USUV co-circulate, the surveillance of WNV allows also the detection of USUV. Due to the great similarity in amino-acid sequence of major surface proteins of the two viruses, a high cross-reactivity can lead to misinterpretation of serological results. Here, we report the results obtained from 54 asymptomatic blood donors during a three-year follow-up showing an unexpected high positivity (46.3%) for USUV. The major obstacle encountered in the differential diagnosis between these two viruses was the high cross-reactivity found in neutralizing antibodies (NT Abs) and, in some cases, a long follow-up was mandatory for a correct diagnosis. Moreover, two new ELISpot assays were developed for a more rapid and specific differential diagnosis, especially in those cases in which NT Abs were not determinant. Using a combination of Enzyme-linked immunospot (ELISpot), molecular, and serological tests, we could identify 25 true positive WNV and 25 true positive USUV blood donors. Our data highlight the importance of raising awareness for increasing USUV infections in endemic countries involved in blood transfusion and organ donation.

Zika virus (ZIKV) infection has been linked to congenital defects in fetuses and infants, as exemplified by the microcephaly epidemic in Brazil. Given the overlapping presence of Dengue virus (DENV) in the majority of ZIKV epidemic regions, advanced diagnostic approaches need to be evaluated to establish the role of pre-existing DENV immunity in ZIKV infection. From 2015 to 2017, five pregnant women with suspected ZIKV infection were investigated in Pavia, Italy. Among the five pregnant women, three were DENV−ZIKV immunologically cross-reactive, and two were DENV-naïve. Advanced diagnosis included the following: (i) NS1 blockade-of-binding (BOB) ELISA assay for ZIKV specific antibodies and (ii) ELISpot assay for the quantification of effector memory T cells for DENV and ZIKV. These novel assays allowed to distinguish between related flavivirus infections. The three DENV-experienced mothers did not transmit ZIKV to the fetus, while the two DENV-naive mothers transmitted ZIKV to the fetus. Pre-existing immunity in DENV experienced mothers might play a role in cross-protection.

West Nile virus lineage 2 (WNV-2) was mainly confined to sub-Saharan Africa until the early 2000s, when it was identified for the first time in Central Europe causing outbreaks of human and animal infection. The aim of this study was to reconstruct the origin and dispersion of WNV-2 in Central Europe and Italy on a phylodynamic and phylogeographical basis. To this aim, discrete and continuous space phylogeographical models were applied to a total of 33 newly characterised full-length viral genomes obtained from mosquitoes, birds and humans in Northern Italy in the years 2013-2015 aligned with 64 complete sequences isolated mainly in Europe. The European isolates segregated into two highly significant clades: a small one including three sequences and a large clade including the majority of isolates obtained in Central Europe since 2004. Discrete phylogeographical analysis showed that the most probable location of the root of the largest European clade was in Hungary a mean 12.78 years ago. The European clade bifurcated into two highly supported subclades: one including most of the Central/East European isolates and the other encompassing all of the isolates obtained in Greece. The continuous space phylogeographical analysis of the Italian clade showed that WNV-2 entered Italy in about 2008, probably by crossing the Adriatic sea and reaching a central area of the Po Valley. The epidemic then spread simultaneously eastward, to reach the region of the Po delta in 2013, and westward to the border area between Lombardy and Piedmont in 2014; later, the western strain changed direction southward, and reached the central area of the Po valley once again in 2015. Over a period of about seven years, the virus spread all over an area of northern Italy by following the Po river and its main tributaries.

Lineage 2 West Nile virus (WNV) caused a vast epidemic in Europe in 2018, with the highest incidence being recorded in Italy. To reconstruct the evolutionary dynamics and epidemiological history of the virus in Italy, 53 envelope gene and 26 complete genome sequences obtained from human and animal samples were characterised by means of next-generation sequencing. Phylogenetic analysis revealed two Italian strains originating between 2010 and 2012: clade A, which apparently became extinct in 2013−2014, and clade B, which was responsible for the 2018 epidemic. The mean genetic distances in clade B increased over time and with the distance between sampling locations. Bayesian birth-death and coalescent skyline plots of the clade B showed that the effective number of infections and the effective reproduction number (Re) increased between 2015 and 2018. Our data suggest that WNV-2 entered Italy in 2011 as a result of one or a few penetration events. Clade B differentiated mainly as a result of genetic drift and purifying selection, leading to the appearance of multiple locally circulating sub-clades for different times. Phylodynamic analysis showed a current expansion of the infection among reservoir birds and/or vectors.

Because swine influenza virus infection is seldom diagnosed in humans, its frequency might be underestimated. We report a immunocompromised hematologic patient with swine influenza A(H3N2) virus in 2014 in Italy. Local pigs were the source of this human infection.

Primary human cytomegalovirus (HCMV) infections during pregnancy are associated with a high risk of virus transmission to the fetus. To identify correlates of intrauterine HCMV transmission, serial serum samples from HCMV transmitter and non-transmitter pregnant women with primary HCMV infection were analyzed for the presence of neutralizing antibodies against different glycoproteins and glycoprotein complexes, which are known to mediate entry into distinct types of host cells. Neutralizing activity was detected in the sera early after primary infection; absorption with a soluble pentameric complex formed by gH/gL/pUL128-131, but not with gH/gL dimer or with gB, abolished the capacity of sera to neutralize infection of epithelial cells. Importantly, an early, high antibody response to pentamer antigenic sites was associated with a significantly reduced risk of HCMV transmission to the fetus. This association is consistent with the high in vitro inhibition of HCMV infection of epithelial/endothelial cells as well as cell-to-cell spreading and virus transfer to leukocytes by anti-pentamer antibodies. Taken together, these findings indicate that the HCMV pentamer complex is a major target of the antibody-mediated maternal immunity.

Background Human parainfluenza virus type 3 (hPIV-3) has been reported to cause nosocomial outbreaks of respiratory infection, in particular among hematopoietic stem cell transplantation recipients.Design and Methods From September 2007 through January 2008 several episodes of hPIV-3 infection were observed among young patients followed at the Oncohematology Unit (OHU) or other units of the Pediatrics Department. In 32 young patients (median age 3.5 years, range 21 days–27 years), hPIV-3 infection was diagnosed by direct fluorescent antibody staining of cells from respiratory secretions, and virus quantified by real-time RT-PCR in nasopharyngeal aspirates or bronchoalveolar lavage samples. In addition, the epidemiologic relatedness of hPIV-3 strains was investigated by sequencing two variable regions of the hemagglutinin-neuraminidase gene (nt 1–569 and nt 762–1239).Results Of the 32 hPIV-3-positive patients, 19 were hematopoietic stem cell transplantation recipients, 8 had hematologic malignancies, and 5 were immunocompetent children. Sixteen patients had upper, and 16 lower respiratory tract infection. All patients but one had high viral load in nasopharyngeal aspirates (>1.0×106 RNA copies/mL). One patient died from respiratory failure with a high viral load in bronchoalveolar lavage. Phylogenetic analysis showed that 16/32 strains were identical. Besides this major cluster, three other clusters were identified, each one defining a smaller outbreak.Conclusions Phylogenetic analysis allows identification of the role of a single or multiple hPIV-3 strains in the person-to-person transmission within an outbreak occurring in clinical units.