Bacterial Biofilm in Chronic Wounds and Possible Therapeutic Approaches.

Wound repair and skin regeneration is a very complex orchestrated process that is generally composed of four phases: hemostasis, inflammation, proliferation, and remodeling. Each phase involves the activation of different cells and the production of various cytokines, chemokines, and other inflammatory mediators affecting the immune response. The microbial skin composition plays an important role in wound healing. Indeed, skin commensals are essential in the maintenance of the epidermal barrier function, regulation of the host immune response, and protection from invading pathogenic microorganisms. Chronic wounds are common and are considered a major public health problem due to their difficult-to-treat features and their frequent association with challenging chronic infections. These infections can be very tough to manage due to the ability of some bacteria to produce multicellular structures encapsulated into a matrix called biofilms. The bacterial species contained in the biofilm are often different, as is their capability to influence the healing of chronic wounds. Biofilms are, in fact, often tolerant and resistant to antibiotics and antiseptics, leading to the failure of treatment. For these reasons, biofilms impede appropriate treatment and, consequently, prolong the wound healing period. Hence, there is an urgent necessity to deepen the knowledge of the pathophysiology of delayed wound healing and to develop more effective therapeutic approaches able to restore tissue damage. This work covers the wound-healing process and the pathogenesis of chronic wounds infected by biofilm-forming pathogens. An overview of the strategies to counteract biofilm formation or to destroy existing biofilms is also provided.

Dynamics of humoral and cellular response to three doses of anti-SARS-CoV-2 BNT162b2 vaccine in patients with hematological malignancies and older subjects.

Background: Few data are available about the durability of the response, the induction of neutralizing antibodies, and the cellular response upon the third dose of the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in hemato-oncological patients. Objective: To investigate the antibody and cellular response to the BNT162b2 vaccine in patients with hematological malignancy. Methods: We measured SARS-CoV-2 anti-spike antibodies, anti-Omicron neutralizing antibodies, and T-cell responses 1 month after the third dose of vaccine in 93 fragile patients with hematological malignancy (FHM), 51 fragile not oncological subjects (FNO) aged 80-92, and 47 employees of the hospital (healthcare workers, (HW), aged 23-66 years. Blood samples were collected at day 0 (T0), 21 (T1), 35 (T2), 84 (T3), 168 (T4), 351 (T pre-3D), and 381 (T post-3D) after the first dose of vaccine. Serum IgG antibodies against S1/S2 antigens of SARS-CoV-2 spike protein were measured at every time point. Neutralizing antibodies were measured at T2, T3 (anti-Alpha), T4 (anti-Delta), and T post-3D (anti-Omicron). T cell response was assessed at T post-3D. Results: An increase in anti-S1/S2 antigen antibodies compared to T0 was observed in the three groups at T post-3D. After the third vaccine dose, the median antibody level of FHM subjects was higher than after the second dose and above the putative protection threshold, although lower than in the other groups. The neutralizing activity of antibodies against the Omicron variant of the virus was tested at T2 and T post-3D. 42.3% of FHM, 80,0% of FNO, and 90,0% of HW had anti-Omicron neutralizing antibodies at T post-3D. To get more insight into the breadth of antibody responses, we analyzed neutralizing capacity against BA.4/BA.5, BF.7, BQ.1, XBB.1.5 since also for the Omicron variants, different mutations have been reported especially for the spike protein. The memory T-cell response was lower in FHM than in FNO and HW cohorts. Data on breakthrough infections and deaths suggested that the positivity threshold of the test is protective after the third dose of the vaccine in all cohorts. Conclusion: FHM have a relevant response to the BNT162b2 vaccine, with increasing antibody levels after the third dose coupled with, although low, a T-cell response. FHM need repeated vaccine doses to attain a protective immunological response.

Skin dysbiosis and Cutibacterium acnes biofilm in inflammatory acne lesions of adolescents.

Acne vulgaris is a common inflammatory disorder affecting more than 80% of young adolescents. Cutibacterium acnes plays a role in the pathogenesis of acne lesions, although the mechanisms are poorly understood. The study aimed to explore the microbiome at different skin sites in adolescent acne and the role of biofilm production in promoting the growth and persistence of C. acnes isolates. Microbiota analysis showed a significantly lower alpha diversity in inflammatory lesions (LA) than in non-inflammatory (NI) lesions of acne patients and healthy subjects (HS). Differences at the species level were driven by the overabundance of C. acnes on LA than NI and HS. The phylotype IA1 was more represented in the skin of acne patients than in HS. Genes involved in lipids transport and metabolism, as well as potential virulence factors associated with host-tissue colonization, were detected in all IA1 strains independently from the site of isolation. Additionally, the IA1 isolates were more efficient in early adhesion and biomass production than other phylotypes showing a significant increase in antibiotic tolerance. Overall, our data indicate that the site-specific dysbiosis in LA and colonization by virulent and highly tolerant C. acnes phylotypes may contribute to acne development in a part of the population, despite the universal carriage of the microorganism. Moreover, new antimicrobial agents, specifically targeting biofilm-forming C. acnes, may represent potential treatments to modulate the skin microbiota in acne.

Immunogenicity of three doses of anti-SARS-CoV-2 BNT162b2 vaccine in psoriasis patients treated with biologics.

Introduction: Psoriasis has not been directly linked to a poor prognosis for COVID-19, yet immunomodulatory agents used for its management may lead to increased vulnerability to the dangerous complications of SARS-CoV-2 infection, as well as impair the effectiveness of the recently introduced vaccines. The three-dose antibody response trend and the safety of BNT162b2 mRNA vaccine in psoriasis patients treated with biologic drugs have remained under-researched. Materials and methods: Forty-five psoriatic patients on biologic treatment were enrolled to evaluate their humoral response to three doses of BNT162b2. IgG titers anti-SARS-CoV-2 spike protein were evaluated at baseline (day 0, first dose), after 3 weeks (second dose), four weeks post-second dose, at the time of the third dose administration and 4 weeks post-third dose. Seropositivity was defined as IgG ≥15 antibody-binding units (BAU)/mL. Data on vaccine safety were also collected by interview at each visit. Results: A statistically significant increase in antibody titers was observed after each dose of vaccine compared with baseline, with no significant differences between patients and controls. Methotrexate used in combination with biologics has been shown to negatively influence the antibody response to the vaccine. On the contrary, increasing body mass index (BMI) positively influenced the antibody response. No adverse effects were reported, and no relapses of psoriasis were observed in the weeks following vaccine administration in our study population. Conclusions: Our data are largely consistent with the recent literature on this topic confirming the substantial efficacy and safety of BNT162b2 mRNA vaccine on psoriatic patients treated with biologics of different types and support the recommendation to perform additional doses in this specific subgroup of patients.

Immunogenicity and Safety of BNT162b2 Homologous Booster Vaccination in People Living with HIV under Effective cART.

Data on COVID-19 boosting vaccination in people living with HIV (PLWH) are scant. We investigated the immunogenicity and safety of the BNT162b2 homologous boosting vaccination. Anti-SARS-CoV-2 spike antibodies (LIAISON® SARS-CoV-2 S1/S2 IgG test, DiaSorin®), CD4+, CD8+ and viraemia were monitored at T0 (pre-vaccination), T1 (4 weeks after the second dose), T2 (pre-booster) and T3 (4 weeks after the booster dose). Humoral responses were evaluated according to sex, age, BMI, nadir and baseline CD4+ counts, as well as type of cART regimen. Forty-two subjects were included: the median age was 53 years (IQR: 48−61); the median time since HIV was 12.4 years (IQR: 6.5−18.3); the median nadir and baseline CD4+ counts were 165 (IQR: 104−291) and 687 cells/mm3 (IQR: 488−929), respectively. The booster dose was administered at a median of 5.5 months after the second dose. Median anti-SARS-CoV-2 IgG concentration had significantly decreased at T2 compared to T1 (107 vs. 377, p < 0.0001). Antibody levels elicited by the booster dose (median: 1580 AU/mL) were significantly higher compared with those of all the other time points (p < 0.0001). None of the investigated variables significantly affected antibody response induced by the booster dose. Local and systemic side-effects were referred by 23.8% and 14.3% of the subjects, respectively. One patient developed sensorineural hearing loss (SNHL) 24 h after boosting. He recovered auditory function upon endothympanic administration of corticosteroids. The BNT162b2 boosting vaccination in PLWH is safe and greatly increased the immune response with respect to the primary vaccination.

Optimizing the Illumina COVIDSeq laboratorial and bioinformatics pipeline on thousands of samples for SARS-CoV-2 Variants of Concern tracking.

The SARS-CoV-2 Variants of Concern tracking via Whole Genome Sequencing represents a pillar of public health measures for the containment of the pandemic. The ability to track down the lineage distribution on a local and global scale leads to a better understanding of immune escape and to adopting interventions to contain novel outbreaks. This scenario poses a challenge for NGS laboratories worldwide that are pressed to have both a faster turnaround time and a high-throughput processing of swabs for sequencing and analysis. In this study, we present an optimization of the Illumina COVID-seq protocol carried out on thousands of SARS-CoV-2 samples at the wet and dry level. We discuss the unique challenges related to processing hundreds of swabs per week such as the tradeoff between ultra-high sensitivity and negative contamination levels, cost efficiency and bioinformatics quality metrics.

Evidence of a SARS-CoV-2 double Spike mutation D614G/S939F potentially affecting immune response of infected subjects.

OBJECTIVES: Despite extensive efforts to monitor the diffusion of COVID-19, the actual wave of infection is worldwide characterized by the presence of emerging SARS-CoV-2 variants. The present study aims to describe the presence of yet undiscovered SARS-CoV-2 variants in Italy. METHODS: Next Generation Sequencing was performed on 16 respiratory samples from occasionally employed within the Bangladeshi community present in Ostia and Fiumicino towns. Computational strategy was used to identify all potential epitopes for reference and mutated Spike proteins. A simulation of proteasome activity and the identification of possible cleavage sites along the protein guided to a combined score involving binding affinity, peptide stability and T-cell propensity. RESULTS: Retrospective sequencing analysis revealed a double Spike D614G/S939F mutation in COVID-19 positive subjects present in Ostia while D614G mutation was evidenced in those based in Fiumicino. Unlike D614G, S939F mutation affects immune response by the slight but significant modulation of T-cell propensity and the selective enrichment of potential binding epitopes for some HLA alleles. CONCLUSION: Collectively, our findings mirror further the importance of deep sequencing of SARS-CoV-2 genome as a unique approach to monitor the appearance of specific mutations as for those herein reported for Spike protein. This might have implications on both the type of immune response triggered by the viral infection and the severity of the related illness.

Antibody Persistence 6 Months Post-Vaccination with BNT162b2 among Health Care Workers.

BACKGROUND: We present immunogenicity data 6 months after the first dose of BNT162b2 in correlation with age, gender, BMI, comorbidities and previous SARS-CoV-2 infection. METHODS: An immunogenicity evaluation was carried out among health care workers (HCW) vaccinated at the Istituti Fisioterapici Ospitalieri (IFO). All HCW were asked to be vaccine by the national vaccine campaign at the beginning of 2021. Serum samples were collected on day 1 just prior to the first dose of the vaccine and on day 21 just prior to the second vaccination dose. Thereafter sera samples were collected 28, 49, 84 and 168 days after the first dose of BNT162b2. Quantitative measurement of IgG antibodies against S1/S2 antigens of SARS-CoV-2 was performed with a commercial chemiluminescent immunoassay. RESULTS: Two hundred seventy-four HWCs were analyzed, 175 women (63.9%) and 99 men (36.1%). The maximum antibody geometric mean concentration (AbGMC) was reached at T2 (299.89 AU/mL; 95% CI: 263.53-339.52) with a significant increase compared to baseline (p < 0.0001). Thereafter, a progressive decrease was observed. At T5, a median decrease of 59.6% in COVID-19 negative, and of 67.8% in COVID-19 positive individuals were identified with respect to the highest antibody response. At T1, age and previous COVID-19 were associated with differences in antibody response, while at T2 and T3 differences in immune response were associated with age, gender and previous COVID-19. At T4 and T5, only COVID-19 positive participants demonstrated a greater antibody response, whereas no other variables seemed to influence antibody levels. CONCLUSIONS: Overall our study clearly shows antibody persistence at 6 months, albeit with a certain decline. Thus, the use of this vaccine in addressing the COVID-19 pandemic is supported by our results that in turn open debate about the need for further boosts.

Immunogenicity and Safety of COVID-19 Vaccine BNT162b2 for Patients with Solid Cancer: A Large Cohort Prospective Study from a Single Institution.

PURPOSE: We assessed the immunogenicity and safety of the BNT162b2 vaccine in a large cohort of patients with cancer (CP). EXPERIMENTAL DESIGN: From March 1, 2021 to March 20, 2021, this prospective cohort study included 816 CP afferent to our institution and eligible for the vaccination. A cohort of 274 health care workers (HCW) was used as age- and sex-matched control group. BNT162b2 was administered as a two-dose regimen given 21 days apart. Blood samples to analyze anti-Spike (S) IgG antibodies (Ab) were collected prevaccination [timepoint (TP) 0], and at 3 weeks (TP1) and 7 weeks (TP2) after the first dose. RESULTS: Patients characteristics: median age 62 (range, 21-97); breast/lung cancer/others (31/21/48%); active treatment/follow-up (90/10%). In the whole CP cohort, the serologic response rate (RR) and the titre of anti-S IgG significantly increased across the TPs; at TP2, the responders (IgG >15 AU/mL) were 94.2%. Active chemotherapy and chronic use of steroids were independent predictors of lower RR. Adverse events (AE) after the booster predicted higher likelihood of response (OR, 4.04; 95% confidence interval, 1.63-9.99; P = 0.003). Comparing the matched cohorts, the responders were significantly lower in CP than in HCW at TP1 (61.2% vs. 93.2%) and TP2 (93.3% vs. 100%), while the geometric mean concentration of IgG did not significantly differ at TP2 being significantly lower in CP (23.3) than in HCW (52.1) at TP1. BNT162b2 was well tolerated in CP; severe-grade AEs were 3.5% and 1.3% after the first and second doses, respectively. CONCLUSIONS: BNT162b2 assures serologic immunization without clinically significant toxicity in CP. The second dose is needed to reach a satisfactory humoral response.

Early Onset of SARS-COV-2 Antibodies after First Dose of BNT162b2: Correlation with Age, Gender and BMI.

BACKGROUND: The first goal of the study was to analyse the antibody titre 21 days after the first dose of the BNT162b2 vaccine in a group of 252 healthcare workers (HCW). The second goal was to analyse how the antibody titre changes in correlation with age, gender and body mass index (BMI). METHODS: Participants had a nasopharyngeal swab for SARS-CoV-2 and were assessed for the presence of SARS-CoV-2 antibodies at baseline and 21 days after the BNT162b2 priming dose. RESULTS: First dose of BNT162b2 activated immune responses in 98% of the participants. Five HWC had no increase in antibody titre 21 days after the first dose. Antibody titre was greater in young (<38 years) vs. older participants (<38 vs. 47-56 p = 0.002; <38 vs. >56 p = 0.001). Higher antibody levels were detected in underweight vs. pre-obesity group (p = 0.026) and in normal-weight vs. pre-obesity group (p = 0.007). This association was confirmed after adjusting for age (p = 0.0001) and gender (p = 0.00001). CONCLUSIONS: Our study demonstrates that a single dose of BNT162b2 activates the immune response, and being young and normal-weight correlate positively with this response. Larger specifically designed clinical trials are needed to validate these results.

Lower response to BNT162b2 vaccine in patients with myelofibrosis compared to polycythemia vera and essential thrombocythemia.

In a population of 42 Philadelphia negative myeloproliferative neoplasm patients, all on systemic active treatment, the likelihood of responding to anti-SARS-CoV-2 BNT162b2 vaccine at 2 weeks after the second dose was significantly lower in the ten patients with myelofibrosis compared to the 32 with essential thrombocythemia (n = 17) and polycythemia vera (n = 15) grouped together, both in terms of neutralizing anti-SARS-CoV-2 IgG titers and seroprotection rates (32.47 AU/mL vs 217.97 AU/mL, p = 0.003 and 60% vs 93.8%, p = 0.021, respectively). Ruxolitinib, which was the ongoing treatment in five patients with myelofibrosis and three with polycythemia vera, may be implicated in reducing vaccine immunogenicity (p = 0.076), though large prospective study is needed to address this issue.

Initial observations on age, gender, BMI and hypertension in antibody responses to SARS-CoV-2 BNT162b2 vaccine.

BACKGROUND: Literature data suggests that age, gender and body mass index (BMI) could be associated with difference in immune responses to vaccines. The first goal of the study was to analyze the antibody titre seven days after the second dose of BNT162b2 vaccine in a group of 248 healthcare workers (HCWs). The second goal was to analyze how antibody titre changes in correlation with age, gender, BMI and hypertension. METHODS: An immunogenicity evaluation was carried out among HCWs vaccinated at the Istituti Fisioterapici Ospitalieri (IFO), Rome, Italy. All HCWs were asked to be vaccinated by the Italian national vaccine campaign at the beginning of 2021. 260 vaccinated HCWs were enrolled in the study. All eligible participants were assigned to receive the priming dose in two weeks' time and the booster dose exactly 21 days thereafter. Blood and nasopharyngeal swabs were collected at baseline and 7 days after second dose of vaccine. Quantitative measurements of IgG antibodies against S1/S2 antigens of SARS-CoV-2 were performed with a commercial chemiluminescent immunoassay. Presence of SARS-Cov-2 in nasopharyngeal swab was determined by commercial RT-PCR testing. FINDINGS: 248 HWCs were analyzed, 158 women (63.7%) and 90 men (36.3%). After the second dose of BNT162b2 vaccine, 99.5% of participants developed a humoral immune response. The geometric mean concentration of antibodies among the vaccinated subjects after booster dose (285.9 AU/mL 95% CI: 249.5-327.7) was higher than that of human convalescent sera (39.4 AU/mL, 95% CI: 33.1-46.9), with p<0.0001. Multivariate linear regression analysis of AU/mL by age, gender and BMI multivariate was performed by the inclusion of covariates. This analysis demonstrated that age (p<0.0001) and gender (p = 0.038) are statistically associated with differences in antibody response after vaccination, whereas BMI and hypertension have no statistically significant association (p = 0.078 and p = 0.52 respectively). INTERPRETATION: 99.5% of HCW developed a humoral immune response and female and young participants seem to have an increased capacity to mount humoral immune responses. BMI and hypertension seem not associated with difference in immune response to the vaccine. FUNDING: None.

Short Communication: HIV Viral Load Trends During the Coronavirus Disease 2019 Pandemic in a Reference Center for HIV in Rome, Italy.

Coronavirus disease 2019 (COVID-19) pandemic has reduced the access of HIV patients to reference centers. However, retention-in-care is critical to maintain adherence to therapy and viral suppression. During lockdown in Italy, our center implemented several measures to ensure HIV-care continuum. To assess whether these efforts were successful, we investigated HIV viral load trend for a 1-year period (September 2019-August 2020), which included lockdown and partial lockdown months in our country. No significant changes overtime in the proportion of undetectable HIV-RNA were observed. Continuity of service made it possible to maintain viral suppression in our patients.

Fifth-week immunogenicity and safety of anti-SARS-CoV-2 BNT162b2 vaccine in patients with multiple myeloma and myeloproliferative malignancies on active treatment: preliminary data from a single institution.

BACKGROUND: Safety and immunogenicity of BNT162b2 mRNA vaccine are unknown in hematological patients; both were evaluated prospectively in 42 patients with multiple myeloma (MM) and 50 with myeloproliferative malignancies (MPM) (20 chronic myeloid leukemias and 30 myeloproliferative neoplasms), all of them on active anti-cancer treatment, in comparison with 36 elderly controls not suffering from cancer. Subjects serologically and/or molecularly (by nasal/throat swab) positives at basal for SARS-CoV-2 were excluded. Primary endpoint was to compare titers of neutralizing anti-SARS-CoV-2 IgG and seroprotection rates among the cohorts at 3 and 5 weeks from first dose. METHODS: Titration was done using LIAISON® SARS-CoV-2 S1/S2 IgG test, a quantitative chemiluminescent immunoassay approved by FDA on the basis of robust evidences of concordance (94.4%) between the test at cutoff of 15 AU/mL and the Plaque Reduction Neutralization Test 90% at 1:40 ratio. Cutoff of 15 AU/mL was assumed to discriminate responders to vaccination with a protective titer. Cohorts were compared using Fisher' exact test and the Mann-Whitney test as appropriated. Geometric mean concentrations (GMCs), geometric mean ratios and response rates after 1st and 2nd dose were compared in each cohort by Wilcoxon and McNemar tests, respectively. RESULTS: At 5 weeks, GMC of IgG in elderly controls was 353.3 AU/mL versus 106.7 in MM (p = 0.003) and 172.9 in MPM patients (p = 0.049). Seroprotection rate at cutoff of 15 AU/mL was 100% in controls compared to 78.6% in MM (p = 0.003) and 88% in MPM patients (p = 0.038). In terms of logarithm of IgG titer, in a generalized multivariate linear model, no gender effect was observed (p = 0.913), while there was a significant trend toward lower titers by increasing age (p < 0.001) and in disease cohorts with respect to controls (MM: p < 0.001 and MPM: p < 0.001). An ongoing treatment without daratumumab was associated with higher likelihood of response in MM patients (p = 0.003). No swabs resulted positive on each time point. No safety concerns were observed. CONCLUSIONS: BNT162b2 has demonstrated to be immunogenic at different extent among the cohorts. Response was 88% and robust in MPM patients. MM patients responded significantly less, particularly those on anti-CD38-based treatment. These latter patients should be advised to maintain masks and social distancing regardless of vaccination status, and their cohabiting family members need to be vaccinated in order to reduce the risk of contagion from the family. Additional boosters and titer monitoring could be considered. Trial registration Study was formally approved by the IRCCS Central Ethical Committee of Regione Lazio in January 2021 (Prot. N-1463/21).

Nucleic Acid Sensing Perturbation: How Aberrant Recognition of Self-Nucleic Acids May Contribute to Autoimmune and Autoinflammatory Diseases.

Bacteria and mammalian cells have developed sophisticated sensing mechanisms to detect and eliminate foreign genetic material or to restrict its expression and replication. Progress has been made in the understanding of these mechanisms, which keep foreign or unwanted nucleic acids in check. The complex of mechanisms involved in RNA and DNA sensing is part of a system which is now appreciated as "immune sensing of nucleic acids" or better "nucleic acid immunity." Nucleic acids, which are critical components for inheriting genetic information in all species, including pathogens, are key structures recognized by the innate immune system. However, while nucleic acid recognition is required for host defense against pathogens, there is a potential risk of self-nucleic acids recognition. In fact, besides its essential contribution to antiviral or microbial defense and restriction of endogenous retro elements, deregulation of nucleic acid immunity can also lead to human diseases due to erroneous detection and response to self-nucleic acids, causing sterile inflammation and autoimmunity. In this review we will discuss the roles of nucleic acid receptors in guarding against pathogen invasion, and how the microbial environment could interfere or influence immune sensing in discriminating between self and non-self and how this may contribute to autoimmunity or inflammatory diseases.

The Emerging Role of Microbial Biofilm in Lyme Neuroborreliosis.

Lyme borreliosis (LB) is the most common tick-borne disease caused by the spirochete Borrelia burgdorferi in North America and Borrelia afzelii or Borrelia garinii in Europe and Asia, respectively. The infection affects multiple organ systems, including the skin, joints, and the nervous system. Lyme neuroborreliosis (LNB) is the most dangerous manifestation of Lyme disease, occurring in 10-15% of infected individuals. During the course of the infection, bacteria migrate through the host tissues altering the coagulation and fibrinolysis pathways and the immune response, reaching the central nervous system (CNS) within 2 weeks after the bite of an infected tick. The early treatment with oral antimicrobials is effective in the majority of patients with LNB. Nevertheless, persistent forms of LNB are relatively common, despite targeted antibiotic therapy. It has been observed that the antibiotic resistance and the reoccurrence of Lyme disease are associated with biofilm-like aggregates in B. burgdorferi, B. afzelii, and B. garinii, both in vitro and in vivo, allowing Borrelia spp. to resist to adverse environmental conditions. Indeed, the increased tolerance to antibiotics described in the persisting forms of Borrelia spp., is strongly reminiscent of biofilm growing bacteria, suggesting a possible role of biofilm aggregates in the development of the different manifestations of Lyme disease including LNB.