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

PMN-MDSC frequency discriminates active versus latent tuberculosis and could play a role in counteracting the immune-mediated lung damage in active disease

: Tuberculosis (TB), due to Mycobacterium tuberculosis infection, is still the principal cause of death caused by a single infectious agent. The balance between the bacillus and host defense mechanisms reflects the different manifestations of the pathology. Factors defining this variety are unclear and likely involve both mycobacterial and immunological components. Myeloid derived suppressor cells (MDSC) have been shown to be expanded during TB, but their role in human TB pathogenesis is not clear. We evaluated the frequency of circulating MDSC by flow-cytometry in 19 patients with active TB, 18 with latent TB infection (LTBI), and 12 healthy donors (HD) as control. Moreover, we investigated the capacity of MDSC to modulate the mycobactericidal activity of monocytes. The association between MDSC level and TB chest X-ray severity score was analyzed. We observed that, unlike active TB, polymorphonuclear (PMN)-MDSC are not expanded in LTBI patients, and, by performing a receiver operating characteristic (ROC) curve analysis, we found that PMN-MDSC frequency supported the discrimination between active disease and LTBI. Interestingly, we observed an association between PMN-MDSC levels and the severity of TB disease evaluated by chest X-ray. Specifically, PMN-MDSC frequency was higher in those classified with a low/mild severity score compared to those classified with a high severity score. Moreover, PMN-MDSC can impact mycobacterial growth by inducing ROS production in Bacillus Calmette et Guerin (BCG)-infected monocytes. This effect was lost when tested with M. tuberculosis (MTB), In conclusion, our data indicate that the elevated frequency of PMN-MDSC in IGRA-positive individuals is associated with active TB. Our findings also pointed out a beneficial role of PMN-MDSC during human active TB, most likely associated with the limitation of inflammation-induced tissue damage

Vitamin D as Modulator of Drug Concentrations: A Study on Two Italian Cohorts of People Living with HIV Administered with Efavirenz

To date, vitamin D seems to have a significant role in affecting the prevention and immunomodulation in COVID-19 disease. Nevertheless, it is important to highlight that this pro-hormone has other several activities, such as affecting drug concentrations, since it regulates the expression of cytochrome P450 (CYP) genes. Efavirenz (EFV) pharmacokinetics is influenced by CYPs, but no data are available in the literature concerning the association among vitamin D levels, seasonality (which affects vitamin D concentrations) and EFV plasma levels. For this reason, the aim of this study was to evaluate the effect of 25-hydroxy vitamin D (25(OH)D3) levels on EFV plasma concentrations in different seasons. We quantified 25(OH)D3 by using chemiluminescence immunoassay, whereas EFV plasma concentrations were quantified with the HPLC–PDA method. A total of 316 patients were enrolled in Turin and Rome. Overall, 25(OH)D3levels resulted in being inversely correlated with EFV concentrations. Some patients with EFV levels higher than 4000 ng/mL showed a deficient 25(OH)D3 concentration in Turin and Rome cohorts and together. EFV concentrations were different in patients without vitamin D supplementation, whereas, for vitamin D-administered individuals, no difference in EFV exposure was present. Concerning seasonality, EFV concentrations were associated with 25(OH)D3 deficiency only in winter and in spring, whereas a significant influence was highlighted for 25(OH)D3 stratification for deficient, insufficient and sufficient values in winter, spring and summer. A strong and inverse association between 25(OH)D3and EFV plasma concentrations was suggested. These data suggest that vitamin D is able to affect drug exposure in different seasons; thus, the achievement of the clinical outcome could be improved by also considering this pro-hormone

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

Neutralizing activity and T Cell response after bivalent fifth dose of mRNA vaccine in person living with HIV

OBJECTIVES: To investigate immunogenicity of SARS-CoV-2 vaccine third booster (3BD; fifth dose) with bivalent vaccine original/BA4/5 vaccine in people living with HIV (PLWH). STUDY DESIGN: This is an observational cohort study to evaluate the outcomes of SARS-CoV-2 vaccination (HIV-VAC study). We analyzed microneutralization assay and IFN-γ production in 48 PLWH on ART with CD4 count <200 cell/mm3 and/or previous AIDS according to immunization status: vaccinated PLWH who had a previous SARS-CoV-2 infection (hybrid immunization, HI) vs. those only vaccinated (non-hybrid immunization, nHI) and current CD4 count RESULTS: After 15 days from its administration (T1), the 3BD bivalent mRNA vaccine elicited a statistically significant increase of neutralizing antibodies (nAbs) geometric mean titers (GMTs) from T0 to T1 against W-D614G (fold-increase 4.8; p<0.0001), BA.5 (8.6 p<0.0001), BQ.1.1 (6.4, p<0.0001) and XBB.1 (6.5, p<0.0001). When compared to BA.5, nAbs GMTs against BQ.1.1 and XBB.1 decreased by 3.5 and 4.1-fold, respectively. After controlling for age, years from AIDS diagnosis, CD4 count at administration and CD4 count nadir, the fold change reduction in nAbs response to other VoCs as compared to BA.1, was larger in participants with HI vs. those nHI: 0.59 lower (95%CI 0.36, 0.97, p=0.04) for BQ.1.1 and 0.67 lower (95% CI: 0.47, 0.96, p=0.03) for XBB.1.In contrast, the analysis carried little evidence for an association between current CD4 count and response to the fifth dose of bivalent vaccine. Furthermore, cell-mediated immunity remained stable. CONCLUSIONS: Our data support the current recommendation of offering bivalent mRNA vaccine booster doses to PLWH with low CD4 count or previous AIDS at first vaccination, especially in those who never previously acquired SARS CoV2 and regardless of current CD4 count

Rapid ART initiation with bictegravir/emtricitabine/tenofovir alafenamide in individuals presenting with advanced HIV disease (Rainbow study)

Background: A rapid ART initiation approach can be beneficial in people with advanced HIV disease, in consideration of their high morbidity and mortality. The aim of our study was to evaluate the feasibility, efficacy and safety of rapid ART start with BIC/FTC/TAF in this setting. Methods: Pilot, single-centre, single-arm, prospective, phase IV clinical trial conducted in a tertiary Italian hospital. Thirty ART-naive people presenting with advanced HIV-1 diagnosis (defined as the presence of an AIDS-defining event and/or CD4 cell count 200 cells/mu L at w 48. Two participants developed IRIS and one was diagnosed with disseminated TB and needed an ART switch. Interpretations: Our results support the feasibility, efficacy and safety of BIC/FTC/TAF as a rapid ART strategy in patients with advanced HIV disease. (c) 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/

Long Term Assessment of Anti-SARS-CoV-2 Immunogenicity after mRNA Vaccine in Persons Living with HIV

(1) Background: Waning of neutralizing and cell-mediated immune response after the primary vaccine cycle (PVC) and the first booster dose (BD) is of concern, especially for PLWH with a CD4 count 500/mm(3)). Mixed models were used to compare mean responses over T1-T4 across CD4 groups. (3) Results: 314 PLWH on ART (LCD4 = 56; ICD4 = 120; HCD4 = 138) were enrolled. At T2, levels of nAbs were significantly lower in LCD4 vs. ICD4/HCD4 (p = 0.04). The BD was crucial for increasing nAbs titers above 1:40 at T3 and up to T4 for WD614G. A positive T cell response after PVC was observed in all groups, regardless of CD4 (p = 0.31). (4) Conclusions: Waning of nAbs after PVC was more important in LCD4 group. The BD managed to re-establish higher levels of nAbs against WD614G, which were retained for 5 months, but for shorter time for Omicron BA.1. The T cellular response in the LCD4 group was lower than that seen in participants with higher CD4 count, but, importantly, it remained above detectable levels over the entire study period

Long Term Assessment of Anti-SARS-CoV-2 Immunogenicity after mRNA Vaccine in Persons Living with HIV

(1) Background: Waning of neutralizing and cell-mediated immune response after the primary vaccine cycle (PVC) and the first booster dose (BD) is of concern, especially for PLWH with a CD4 count ≤200 cells/mm3. (2) Methods: Neutralizing antibodies (nAbs) titers by microneutralization assay against WD614G/Omicron BA.1 and IFNγ production by ELISA assay were measured in samples of PLWH at four time points [2 and 4 months post-PVC (T1 and T2), 2 weeks and 5 months after the BD (T3 and T4)]. Participants were stratified by CD4 count after PVC (LCD4, ≤200/mm3; ICD4, 201–500/mm3, and HCD4, &gt;500/mm3). Mixed models were used to compare mean responses over T1–T4 across CD4 groups. (3) Results: 314 PLWH on ART (LCD4 = 56; ICD4 = 120; HCD4 = 138) were enrolled. At T2, levels of nAbs were significantly lower in LCD4 vs. ICD4/HCD4 (p = 0.04). The BD was crucial for increasing nAbs titers above 1:40 at T3 and up to T4 for WD614G. A positive T cell response after PVC was observed in all groups, regardless of CD4 (p = 0.31). (4) Conclusions: Waning of nAbs after PVC was more important in LCD4 group. The BD managed to re-establish higher levels of nAbs against WD614G, which were retained for 5 months, but for shorter time for Omicron BA.1. The T cellular response in the LCD4 group was lower than that seen in participants with higher CD4 count, but, importantly, it remained above detectable levels over the entire study period

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

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 &gt;500&nbsp;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&nbsp;=&nbsp;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&nbsp;=&nbsp;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&nbsp;-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&nbsp;=&nbsp;0.037; OR 0.30 (0.10-0.88), p&nbsp;=&nbsp;0.029 and OR 0.19 (0.05-0.73), p&nbsp;=&nbsp;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&nbsp;×&nbsp;1000, 2021" and by the Italian Ministry of Health "Ricerca Corrente Linea 2"