Potential role of p53 protein as a novelbiomarker of sperm quality, able to predict thesuccess of ART techniques. EcoFoodFertility Project

Introduction: Protein p53 is well known as “The guardian of genome”; it changes its concentration in human spermatozoa DNAin relation to the damage of the latter. It has been suggested thatthe role of the p53 ancestral gene was to ensure the integrity ofthe genomic germline and the fidelity of the evelopment process.The aim of this study is to evaluate if different concentrations of p53 protein in human spermatozoa could influence embryo quality and pregnancy rate and possibly representing a potential predictive marker of sperm quality for successful fertilization .Methods: From July 2013 to June 2017 we have examinatedretrospectively 79 couples with 2-5 years of infertility history.Males had an average age of 27 ± 7,5 years, sperm concentrationof 33,8 ± 6,2 mil/ml, progressive motility of 41,4 ± 8,3 and a typical morphology of 16,5 ± 3,5 according to Kruger’s method. We have divided the couples on the basis of p53 levels: Group A:0,35–1,65 ng/mil (21 males); Group B: 1,66–3,57 ng/mil (32 males);Group C: 3,58–14,53 ng/mil (26 males). We have evaluated thenumber of embryos at stage of 6–8 cells, btained at the third dayof embryo development, in these three different group. In order toevaluate the concentration of p53 protein, we first proceeded toa DNA extraction with forensic method and then to a quantification p53 protein with ELISA-immunoenzymatic assay, expressedin ng/million of spermatozoa.Results: We have observed different percentage of embryo development at stage of 6-8 cells in the third day and different pregnancy rate (PR):Group A: 101 embryos at 6-8 cells/ 147 total number of obtained embryos in this group (68,4%) and PR = 52,38%.Group B: 128/240 (53,5%); PR = 37,50%; Group C: 79/216 (36,1%);PR = 7,69%. These results support the hypothesis that an high con-centration of p53 in human sperm DNA is associated to a low percentage of embryos able to reach the stage of 6-8 cells in the third day of development and also to a lower pregnancy rate. So p53 levels can be considered as a predictive value to embryo development and pregnancy rate.Conclusions: Protein p53 is a sequence-specific transcriptionfactor that responds to a wide variety of stress signals (environ-mental insults and bad lifestyle) as we are investigating within theecofoodfertility project. Particularly quantitative research of p53could be considered as a novel biomarker of sperm quality, able topredict the success of ART echniques, and could open a new roadfor infertility diagnosis

Innovative Approach for Human Semen Quality Assessment Based on Volatilomics

The volatilome profile of some biofluids (blood, urine, and human semen) identified by Solid-Phase Microextraction–Gas Chromatography/Mass Spectrometry (SPME-GC/MS) and collected from young men living in two high-pollution areas in Italy, i.e., Land of Fires and Valley of Sacco River, have been coupled to sperm parameters obtained by spermiogram analysis to build general multiple regression models. Panels of volatile organic compounds (VOCs) have been selected to optimize the models and used as predictive variables to estimate the different sperm quality parameters (sperm cell concentration, total and progressive motility/immotile cells, total/head/neck/tail morphology anomalies, semen round cell concentration). The results of the multiple linear regression models based on the different subgroups of data joining VOCs from one/two or three biofluids have been compared. Surprisingly, the models based on blood and urine VOCs have allowed an excellent estimate of spermiogram values, paving the way towards a new method of indirect evaluation of semen quality and preventive screening. The significance of VOCs in terms of toxicity and dangerousness was discussed with the support of chemical databases available online

Small noncoding RNAs and sperm nuclear basic proteins reflect the environmental impact on germ cells

Background: Molecular techniques can complement conventional spermiogram analyses to provide new information on the fertilizing potential of spermatozoa and to identify early alterations due to environmental pollution. Methods: Here, we present a multilevel molecular profiling by small RNA sequencing and sperm nuclear basic protein analysis of male germ cells from 33 healthy young subjects residing in low and high-polluted areas. Results: Although sperm motility and sperm concentration were comparable between samples from the two sites, those from the high-pollution area had a higher concentration of immature/immune cells, a lower protamine/histone ratio, a reduced ability of sperm nuclear basic proteins to protect DNA from oxidative damage, and an altered copper/zinc ratio in sperm. Sperm levels of 32 microRNAs involved in intraflagellar transport, oxidative stress response, and spermatogenesis were different between the two areas. In parallel, a decrease of Piwi-interacting RNA levels was observed in samples from the high-polluted area. Conclusions: This comprehensive analysis provides new insights into pollution-driven epigenetic alterations in sperm not detectable by spermiogram

Dexamethasone Increases Pigment Epithelium-Derived Factor in Perfused Human Eyes

To investigate the effects of dexamethasone (DEX) on pigment epithelium-derived factor (PEDF) cDNA and secreted protein in human trabecular meshwork (TM)

Comparative analysis of the bioaccumulation of bisphenol A in the blood serum and follicular fluid of women living in two areas with different environmental impacts

IntroductionBisphenol A (BPA) is a common contaminant widely used in many industrial sectors. Because of its wide use and dispersion, it can be accumulated in living human bodies through both oral assumption and nondietary routes. BPA exhibits hormone-like properties, falling under the class of endocrine disruptors; therefore, it can alter relevant physiological functions. In particular, in women, it can affect folliculogenesis and therefore reproduction, contributing not only to infertility, but also to endometriosis and premature puberty.MethodsWe conducted a multicenter study on 91 women undergoing a first in vitro fertilization (IVF) treatment in the Campania region (Southern Italy). We investigated the presence and concentration of BPA in serum and follicular fluids to assess the effects of airborne BPA contamination. The analysis was conducted on 32 women living in a low environmental impact (LEI) area, from the Sele Valley River and Cilento region, and 59 women living in a high environmental impact (HEI) area, the so-called “Land of Fires”, a highly contaminated territory widely exposed to illegal waste practices.ResultsA higher average BPA content in both blood serum and follicular fluid was revealed in the HEI group when compared with the LEI group. In addition, we revealed higher average BPA content in blood serum than in folliclular fluid in the HEI area, with opposite average content in the two fluids in the LEI zone. In addition, our results also showed a lack of correlation between BPA content in follicular and serum fluids both in the overall population and in the HEI and LEI groups, with peculiar trends in different subsets of women.ConclusionFrom our results, we revealed a heterogeneity in the distribution of BPA content between serum and follicular fluid. Further studies are needed to unravel the bioaccumulation mechanisms of BPA in highly polluted and nonpolluted areas

Immunogenicity and reactogenicity of modified vaccinia Ankara pre-exposure vaccination against mpox according to previous smallpox vaccine exposure and HIV infection. Prospective cohort study

Background: Pre-exposure vaccination with MVA-BN has been widely used against mpox to contain the 2022 outbreak. Many countries have defined prioritized strategies, administering a single dose to those historically vaccinated for smallpox, to achieve quickly adequate coverage in front of low supplies. Using epidemiological models, real-life effectiveness was estimated at approximately 36%-86%, but no clinical trials were performed. Few data on MVA-BN immunogenicity are currently available, and there are no established correlates of protection. Immunological response in PLWH in the context of the 2022 outbreak was also poorly described. Methods: Blood samples were collected from participants eligible for pre-exposure MVA-BN vaccination before (T1) receiving a full course of vaccine (single-dose for vaccine-experienced or smallpox-primed and two-dose for smallpox vaccine-naïve or smallpox non-primed) and one month after the last dose (T2 and T3, respectively). MPXV-specific IgGs were measured by in-house immunofluorescence assay, using 1:20 as screening dilution, MPXV-specific nAbs by 50% plaque reduction neutralization test (PRNT50, starting dilution 1:10), and IFN-γ-producing specific T cells to MVA-BN vaccine, by ELISpot assay. Paired or unpaired t-test and Wilcoxon or Mann-Whitney test were used to analyse IgG and nAbs, and T-cell response, as appropriate. The probability of IgG and nAb response in vaccine-experienced vs. vaccine-naïve was estimated in participants not reactive at T1. The McNemar test was used to evaluate vaccination's effect on humoral response both overall and by smallpox vaccination history. In participants who were not reactive at T1, the proportion of becoming responders one month after full-cycle completion by exposure groups was compared by logistic regression and then analysed by HIV status strata (interaction test). The response was also examined in continuous, and the Average Treatment Effect (ATE) of the difference from baseline to schedule completion according to previous smallpox vaccination was estimated after weighting for HIV using a linear regression model. Self-reports of adverse effects following immunization (AEFIs) were prospectively collected after the first MVA-BN dose (T1). Systemic (S-AEFIs: fatigue, myalgia, headache, GI effects, chills) and local (L-AEFIs: redness, swelling, pain) AEFIs were graded as absent (grade 0), mild (1), moderate (2), or severe (3). The maximum level of severity for S-AEFIs and L-AEFIs ever experienced over the 30 days post-dose by vaccination exposure groups were analysed using a univariable multinomial logistic regression model and after adjusting for HIV status; for each of the symptoms, we also compared the mean duration by exposure group using an unpaired t-test. Findings: Among the 164 participants included, 90 (54.8%) were smallpox vaccine-experienced. Median age was 49 years (IQR 41-55). Among the 76 (46%) PLWH, 76% had a CD4 count >500 cells/μL. There was evidence that both the IgG and nAbs titers increased after administration of the MVA-BN vaccine. However, there was no evidence for a difference in the potential mean change in humoral response from baseline to the completion of a full cycle when comparing primed vs. non-primed participants. Similarly, there was no evidence for a difference in the seroconversion rate after full cycle vaccination in the subset of participants not reactive for nAbs at T1 (p = 1.00 by Fisher's exact test). In this same analysis and for the nAbs outcome, there was some evidence of negative effect modification by HIV (interaction p-value = 0.17) as primed people living with HIV (PLWH) showed a lower probability of seroconversion vs. non-primed, and the opposite was seen in PLWoH. When evaluating the response in continuous, we observed an increase in T-cell response after MVA-BN vaccination in both primed and non-primed. There was evidence for a larger increase when using the 2-dose vs. one-dose strategy with a mean difference of -2.01 log2 (p ≤ 0.0001), after controlling for HIV. No evidence for a difference in the risk of developing any AEFIs of any grade were observed by exposure group, except for the lower risk of grade 2 (moderate) fatigue, induration and local pain which was lower in primed vs. non-primed [OR 0.26 (0.08-0.92), p = 0.037; OR 0.30 (0.10-0.88), p = 0.029 and OR 0.19 (0.05-0.73), p = 0.015, respectively]. No evidence for a difference in symptom duration was also detected between the groups. Interpretation: The evaluation of the humoral and cellular response one month after the completion of the vaccination cycle suggested that MVA-BN is immunogenic and that the administration of a two-dose schedule is preferable regardless of the previous smallpox vaccination history, especially in PLWH, to maximize nAbs response. MVA-BN was safe as well tolerated, with grade 2 reactogenicity higher after the first administration in vaccine-naïve than in vaccine-experienced individuals, but with no evidence for a difference in the duration of these adverse effects. Further studies are needed to evaluate the long-term duration of immunity and to establish specific correlates of protection. Funding: The study was supported by the National Institute for Infectious Disease Lazzaro Spallanzani IRCCS "Advanced grant 5 × 1000, 2021" and by the Italian Ministry of Health "Ricerca Corrente Linea 2"

Immunogenicity and reactogenicity of modified vaccinia Ankara pre-exposure vaccination against mpox according to previous smallpox vaccine exposure and HIV infection: prospective cohort study

BACKGROUND: Pre-exposure vaccination with MVA-BN has been widely used against mpox to contain the 2022 outbreak. Many countries have defined prioritized strategies, administering a single dose to those historically vaccinated for smallpox, to achieve quickly adequate coverage in front of low supplies. Using epidemiological models, real-life effectiveness was estimated at approximately 36%–86%, but no clinical trials were performed. Few data on MVA-BN immunogenicity are currently available, and there are no established correlates of protection. Immunological response in PLWH in the context of the 2022 outbreak was also poorly described. METHODS: Blood samples were collected from participants eligible for pre-exposure MVA-BN vaccination before (T1) receiving a full course of vaccine (single-dose for vaccine-experienced or smallpox-primed and two-dose for smallpox vaccine-naïve or smallpox non-primed) and one month after the last dose (T2 and T3, respectively). MPXV-specific IgGs were measured by in-house immunofluorescence assay, using 1:20 as screening dilution, MPXV-specific nAbs by 50% plaque reduction neutralization test (PRNT50, starting dilution 1:10), and IFN-γ-producing specific T cells to MVA-BN vaccine, by ELISpot assay. Paired or unpaired t-test and Wilcoxon or Mann–Whitney test were used to analyse IgG and nAbs, and T-cell response, as appropriate. The probability of IgG and nAb response in vaccine-experienced vs. vaccine-naïve was estimated in participants not reactive at T1. The McNemar test was used to evaluate vaccination's effect on humoral response both overall and by smallpox vaccination history. In participants who were not reactive at T1, the proportion of becoming responders one month after full-cycle completion by exposure groups was compared by logistic regression and then analysed by HIV status strata (interaction test). The response was also examined in continuous, and the Average Treatment Effect (ATE) of the difference from baseline to schedule completion according to previous smallpox vaccination was estimated after weighting for HIV using a linear regression model. Self-reports of adverse effects following immunization (AEFIs) were prospectively collected after the first MVA-BN dose (T1). Systemic (S-AEFIs: fatigue, myalgia, headache, GI effects, chills) and local (L-AEFIs: redness, swelling, pain) AEFIs were graded as absent (grade 0), mild (1), moderate (2), or severe (3). The maximum level of severity for S-AEFIs and L-AEFIs ever experienced over the 30 days post-dose by vaccination exposure groups were analysed using a univariable multinomial logistic regression model and after adjusting for HIV status; for each of the symptoms, we also compared the mean duration by exposure group using an unpaired t-test. FINDING: Among the 164 participants included, 90 (54.8%) were smallpox vaccine-experienced. Median age was 49 years (IQR 41–55). Among the 76 (46%) PLWH, 76% had a CD4 count >500 cells/μL. There was evidence that both the IgG and nAbs titers increased after administration of the MVA-BN vaccine. However, there was no evidence for a difference in the potential mean change in humoral response from baseline to the completion of a full cycle when comparing primed vs. non-primed participants. Similarly, there was no evidence for a difference in the seroconversion rate after full cycle vaccination in the subset of participants not reactive for nAbs at T1 (p = 1.00 by Fisher's exact test). In this same analysis and for the nAbs outcome, there was some evidence of negative effect modification by HIV (interaction p-value = 0.17) as primed people living with HIV (PLWH) showed a lower probability of seroconversion vs. non-primed, and the opposite was seen in PLWoH. When evaluating the response in continuous, we observed an increase in T-cell response after MVA-BN vaccination in both primed and non-primed. There was evidence for a larger increase when using the 2-dose vs. one-dose strategy with a mean difference of −2.01 log2 (p ≤ 0.0001), after controlling for HIV. No evidence for a difference in the risk of developing any AEFIs of any grade were observed by exposure group, except for the lower risk of grade 2 (moderate) fatigue, induration and local pain which was lower in primed vs. non-primed [OR 0.26 (0.08–0.92), p = 0.037; OR 0.30 (0.10–0.88), p = 0.029 and OR 0.19 (0.05–0.73), p = 0.015, respectively]. No evidence for a difference in symptom duration was also detected between the groups. INTERPRETATION: The evaluation of the humoral and cellular response one month after the completion of the vaccination cycle suggested that MVA-BN is immunogenic and that the administration of a two-dose schedule is preferable regardless of the previous smallpox vaccination history, especially in PLWH, to maximize nAbs response. MVA-BN was safe as well tolerated, with grade 2 reactogenicity higher after the first administration in vaccine-naïve than in vaccine-experienced individuals, but with no evidence for a difference in the duration of these adverse effects. Further studies are needed to evaluate the long-term duration of immunity and to establish specific correlates of protection

Luigi Settembrini. Periodico letterario educativo mensile. A. 2, n.1(1892)-A. 3, n.10(1894)

A.2, n.1(nov.1892): G. Olivieri, Ad Antonio Bartolini, P. 1-2 ; G. Olivieri, Prospero Viani, P. 2-11; R. Sabbatini, Ancora Quom o Quum?, P. 11-2 ; Pensieri del Settembrini, P. 12-3 ; E. Perito, Ad nubem, P. 13; F. Lagrance, Dell’educazione fisica, p. 14-15 ; Cronaca dell’Istituto L. Settembrini, P. 15-6.A. 2, n. 2(dic. 1892): G. Lanzalone, La morale nell’arte, P. 17-23 ; B. ( dal Bibliografo), Per i libri di testo nelle scuole elementari, P. 23-5 ; V. Notari, In Gutembergium artis typograficae inventorem, P. 25; Progressi della navigazione aerea, P. 26-8 ; Pensieri del Settembrini, P. 28-9 ; Il nostro concorso, P. 29 ; G. Lanzalone, La prima pioggia d’autunno, P. 30-1A. 2, n.3(gen. 1893): Bonghi, Una lettera del Bonghi, P. 33-4 ; G. Lanzalone, Ancora della morale nell’arte, P. 34-8 ; Pensieri del Settembrini, P. 38-9 ; C. Arlìa, Buon dì e tre anguille, P. 39-41 ; G. Lanzalone , All’amico G. Olivieri , P. 42-3 ; P. Lioy, Bagni e villeggiature, P. 43-5.A. 2, n. 4(feb. 1893): C. A. Alemagna, Per la morale nell’arte, P. 49-55.A. 2, n. 5(mar. 1893): R. Sabbatini, L’epistola di Saffo a Faone, P. 65-6 ; G. Lanzalone, Il drammaturgo(caricatura), P. 66-7 ; G. Lanzalone, Ancora per la morale dell’arte, P. 67-70 ; F. De Falco, Un’altra lettera!, P. 71-2 ; Il Settembrini, Per una petizione al Parlamento, P. 72; E. Perito, A mia sorella morta, P. 73-4.A. 2, n.6(apr. 1893): Il Settembrini, Petizione al Parlamento, P. 81-3 ; C. Arlia, Noterelle filologiche, P. 84-5 ; G. Bigoni, Ricordi Picentini (2 sonetti), P. 85 ; C. A. Alemagna, Lettera con annotazioni, P. 86-90 ; V. Caputo, Il giuramento, P. 90-1 ; A. Buscaino Campo, Il piè fermo di Dante, P. 92-3.A.2, n.7(apr. 1893): G. Lanzalone, Professori e maestri, P. 97-9 ; il Settembrini, Petizione al Parlamento, P. 100-2 ; Lista delle adesioni, P. 102-4 ; Guido Bigoni, Domenica Rusticana, P. 105 ; Pensieri del Settembrini, P. 105-7 ; Dal Gazzettino d’oro, Utili varietà, P. 107-8 ; G. Lanzalone, Al maggiore Vincenzo Notari, P. 108 ; V. Notarius, Risposta, P. 109 ; F. Accinelli, La poesia della vita, P. 109-10.A. 2, n.8(giu. 1893): C. Arlìa, Noterelle filologiche, P. 113-16 ; Guido Bigoni, La quercia del Tasso, P. 116 ; G. Lanzalone, La ginnastica con la neve, P. 116-17 ; Luigi Settembrini, Una lettera inedita di L. Settembrini, P. 118-19 ; V. Notaro, In Ariostum, P. 119 ; L’arte di respirare, P. 120-21 ; F. De Falco, Il suicidio e la religione, P. 122-24.A.2, n.9/10(lug.-ago. 1893): R. Mariano, Ad un banchetto nunziale, P. 129-132 ; G. Lanzalone, A Cristoforo Colombo, P. 132-34 ; C. Arlìa, Note filologiche, P. 135-36 ; G. Olivieri, Il terzo libro della vita di G. Cristo, P. 136-39 ; F. Persico, La pace, P. 140-41 ; L. A. Villari, Cesare Dalbono, P. 141-45.A.2, n.11/12(sett.-ott. 1893): Luigi Settembrini, Una lettera inedita di L. Settembrini, P. 153-54 ; A. De Leo, Vite di illustri salernitani, P. 154-62 ; G. Franciosi, I sogni, P. 162-63 ; A. Frabasile, Bozzetti ellenici, P. 164-70 ; L. A. Villari , Errori Giudiziari, P. 171-75 ; G. Lanzalone, Verismo, P. 175-76.A.3,n.1/2 (nov.-dic. 1893): M. Giordano, La pubblica educazione e l’ateismo, P. 1-4 ; il Settembrini, Concorso, P. 4 ; Per una forca conservata in un museo, P. 5 ; G. Lanzalone, L’Ora presente, P. 5 ; C. Mariano Pilar, Silvio Spaventa, P. 6-19 ; C. Arlìa, Note filologiche, P. 20-1 ; G. Grammatica, Ideale, P. 22 ; G. Olivieri, Funesta rimembranza, P. 31-2 ; G. Olivieri, La notte della vigilia del Natale, P. 32.A.3, n.3/4(gen.-feb. 1894): G. Lanzalone, E la nostra petizione?, P. 33-5 ; Dall’albo di Luigi Antonio Villari, P. 36 ; G. Olivieri, Una visita inaspettata, P. 37-46 ; A. Frabasile, Alla signora G. P., P. 47 ; M. Giordano, Il Governo, i Municipi e l’istruzione religiosa, P. 48-53 ; G. Lanzalone, Esercizi militari, P. 53 ; C. Arlìa, Note filologiche, P. 54 ; G. Lanzalone, Un dubbio proposto al prof. Sabbadini, P. 54-5 ; G. L., Risultato del passato concorso e concorso nuovo, P. 55-6 ; R. Galdi, Epistola di Catullo ad Ortalo, P. 57.A.3, n.5/6(mar-apr 1894): G. Lanzalone, Dell’educazione nelle scuole classiche, P. 65-70 ; G. L., Un epigramma di Leone XIII, P. 70-1 ; V. Caputo, Vita di borso, P. 72-6 ; C. Arlìa, Note filologiche, P. 76-7 ; G. Lanzalone, Amore, P. 78 ; Aniello Gaeta, Dulcissime Rerum, P. 79-81 ; Francesco De Falco, La duchessa Ravaschieri e il dormitorio, P. 81-2 ; Carmine Zottoli, Risultato del passato concorso e concorso nuovo, P. 82-6.A.3, n. 7/8(mag. – giu 1894): C. Arlìa, Note filologiche, P. 97-8 ; G. Lanzalone, Per il 1°Maggio, P. 98-101 ; M. Giordano, La libertà d’insegnamento e di coscienza, P. 101-10 ; L. A. Villari, Il capitano Tim- Tim, P. 111- 15 ; C. A. Alemagna, L’opera recente di Herbert Spencer, P. 115-16 ; Nicc. Castagna, Sospiro, P. 116 ; G. Cuomo, Sovra un passo del carme “I Sepolcri”, P. 117-20.A.3, n.9(lug. 1894): Concorso nuovo, P. 125 ; C. Arlìa, Note filologiche, P. 126-27 ; Il manicomio dei genii, P. 127 ; G. Lanzalone, Elena, P. 128 ; Giovanni Cuomo, Nunzio del giorno, P. 128-29 ; Giovanni Manfredi, Ofelia, P. 129 ; G. Lanzalone, Da Anacreonte, P. 139.A.3, n.10(ago. 1894): R. Sabbadini, L’anno della nascita di Gasparino Barziza, P. 141-42 ; G. Lanzalone, Il Discredito dei versi, P. 142-46 ; C. Arlìa, Note filologiche, P. 147-48 ; E. Perito, L’ultima rosa d’estate, P. 148 ; D’Aloja, L’arte e la critica, P. 149-51 ; Epigrammi, P. 151-52

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

IntroductionImmunocompromised patients have been shown to have an impaired immune response to COVID-19 vaccines.MethodsHere we compared the B-cell, T-cell and neutralizing antibody response to WT and Omicron BA.2 SARS-CoV-2 virus after the fourth dose of mRNA COVID-19 vaccines in patients with hematological malignancies (HM, n=71), solid tumors (ST, n=39) and immune-rheumatological (IR, n=25) diseases. The humoral and T-cell responses to SARS-CoV-2 vaccination were analyzed by quantifying the anti-RBD antibodies, their neutralization activity and the IFN-γ released after spike specific stimulation.ResultsWe show that the T-cell response is similarly boosted by the fourth dose across the different subgroups, while the antibody response is improved only in patients not receiving B-cell targeted therapies, independent on the pathology. However, 9% of patients with anti-RBD antibodies did not have neutralizing antibodies to either virus variants, while an additional 5.7% did not have neutralizing antibodies to Omicron BA.2, making these patients particularly vulnerable to SARS-CoV-2 infection. The increment of neutralizing antibodies was very similar towards Omicron BA.2 and WT virus after the third or fourth dose of vaccine, suggesting that there is no preferential skewing towards either virus variant with the booster dose. The only limited step is the amount of antibodies that are elicited after vaccination, thus increasing the probability of developing neutralizing antibodies to both variants of virus.DiscussionThese data support the recommendation of additional booster doses in frail patients to enhance the development of a B-cell response directed against Omicron and/or to enhance the T-cell response in patients treated with anti-CD20