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The majority of naturally elicited antibodies against the HIV-1 envelope glycoproteins (Env) are non-neutralizing (nnAbs) because they are unable to recognize the Env trimer in its native "closed" conformation. Nevertheless, it has been shown that nnAbs have the potential to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC) provided that Env is present on the cell surface in its "open" conformation. This is because most nnAbs recognize epitopes that become accessible only after Env interaction with CD4 and the exposure of epitopes that are normally occluded in the closed trimer. HIV-1 limits this vulnerability by downregulating CD4 from the surface of infected cells, thus preventing a premature encounter of Env with CD4. Small CD4-mimetics (CD4mc) sensitize HIV-1-infected cells to ADCC by opening the Env glycoprotein and exposing CD4-induced (CD4i) epitopes. There are two families of CD4i nnAbs, termed anti-cluster A and anti-CoRBS Abs, which are known to mediate ADCC in the presence of CD4mc. Here, we performed Fab competition experiments and found that anti-gp41 cluster I antibodies comprise a major fraction of the plasma ADCC activity in people living with HIV (PLWH). Moreover, addition of gp41 cluster I antibodies to cluster A and CoRBS antibodies greatly enhanced ADCC-mediated cell killing in the presence of a potent indoline CD4mc, CJF-III-288. This cocktail outperformed broadly neutralizing antibodies and even showed activity against HIV-1-infected monocyte-derived macrophages. Thus, combining CD4i antibodies with different specificities achieves maximal ADCC activity, which may be of utility in HIV cure strategies.IMPORTANCEThe elimination of HIV-1-infected cells remains an important medical goal. Although current antiretroviral therapy decreases viral loads below detection levels, it does not eliminate latently infected cells that form the viral reservoir. Here, we developed a cocktail of non-neutralizing antibodies targeting highly conserved Env regions and combined it with a potent indoline CD4mc. This combination exhibited potent ADCC activity against HIV-1-infected primary CD4 + T cells as well as monocyte-derived macrophages, suggesting its potential utility in decreasing the size of the viral reservoir., Competing Interests: The authors declare no conflict of interest.

CD4-mimetics (CD4mcs) are small molecule compounds that mimic the interaction of the CD4 receptor with HIV-1 envelope glycoproteins (Env). Env from primary viruses normally samples a "closed" conformation that occludes epitopes recognized by CD4-induced (CD4i) non-neutralizing antibodies (nnAbs). CD4mcs induce conformational changes on Env resulting in the exposure of these otherwise inaccessible epitopes. Here, we evaluated the capacity of plasma from a cohort of 50 people living with HIV to recognize HIV-1-infected cells and eliminate them by antibody-dependent cellular cytotoxicity (ADCC) in the presence of a potent indoline CD4mc. We observed a marked heterogeneity among plasma samples. By measuring the levels of different families of CD4i Abs, we found that the levels of anti-cluster A, anti-coreceptor binding site, and anti-gp41 cluster I antibodies are responsible for plasma-mediated ADCC in the presence of CD4mc., Importance: There are several reasons that make it difficult to target the HIV reservoir. One of them is the capacity of infected cells to prevent the recognition of HIV-1 envelope glycoproteins (Env) by commonly elicited antibodies in people living with HIV. Small CD4-mimetic compounds expose otherwise occluded Env epitopes, thus enabling their recognition by non-neutralizing antibodies (nnAbs). A better understanding of the contribution of these antibodies to eliminate infected cells in the presence of CD4mc could lead to the development of therapeutic cure strategies., Competing Interests: The authors declare no conflict of interest.

HIV-1 envelope glycoprotein (Env) conformation substantially impacts antibody-dependent cellular cytotoxicity (ADCC). Envs from primary HIV-1 isolates adopt a prefusion "closed" conformation, which is targeted by broadly neutralizing antibodies (bnAbs). CD4 binding drives Env into more "open" conformations, which are recognized by non-neutralizing Abs (nnAbs). To better understand Env-Ab and Env-CD4 interaction in CD4+ T cells infected with HIV-1, we simultaneously measured antibody binding and HIV-1 mRNA expression using multiparametric flow cytometry and RNA flow fluorescent in situ hybridization (FISH) techniques. We observed that env mRNA is almost exclusively expressed by HIV-1 productively infected cells that already downmodulated CD4. This suggests that CD4 downmodulation precedes env mRNA expression. Consequently, productively infected cells express "closed" Envs on their surface, which renders them resistant to nnAbs. Cells recognized by nnAbs were all env mRNA negative, indicating Ab binding through shed gp120 or virions attached to their surface. Consistent with these findings, treatment of HIV-1-infected humanized mice with the ADCC-mediating nnAb A32 failed to lower viral replication or reduce the size of the viral reservoir. These findings confirm the resistance of productively infected CD4+ T cells to nnAbs-mediated ADCC and question the rationale of immunotherapy approaches using this strategy., Importance: Antibody-dependent cellular cytotoxicity (ADCC) represents an effective immune response for clearing virally infected cells, making ADCC-mediating antibodies promising therapeutic candidates for HIV-1 cure strategies. Broadly neutralizing antibodies (bNAbs) target epitopes present on the native "closed" envelope glycoprotein (Env), while non-neutralizing antibodies (nnAbs) recognize epitopes exposed upon Env-CD4 interaction. Here, we provide evidence that env mRNA is predominantly expressed by productively infected cells that have already downmodulated cell-surface CD4. This indicates that CD4 downmodulation by HIV-1 precedes Env expression, making productively infected cells resistant to ADCC mediated by nnAbs but sensitive to those mediated by bnAbs. These findings offer critical insights for the development of immunotherapy-based strategies aimed at targeting and eliminating productively infected cells in people living with HIV.

Trazodone is a triazolpyridine derivative approved for the treatment of depression, and currently marketed as oral formulations. The transdermal administration of this drug could reduce side effects, related to peak plasma concentration, and improve patient adherence due to a reduced administration frequency. The aims of this work were: (a) the evaluation of the effect of pH vehicle and permeation enhancers on trazodone permeability across porcine skin ex-vivo; (b) the development and optimization of a transdermal drug delivery system containing trazodone hydrochloride. From the results obtained, it was found that the effect of pH of the vehicle on the permeation of trazodone across the skin is quite complex, because it influences both solubility and partitioning and that the presence of fatty acids in the vehicle has a notable effect on permeation (the enhancement factor obtained was approx. 100). For both the fatty acid selected (oleic and lauric) a parabolic relationship between the transdermal flux and the concentration was found, with an optimum activity in the range 2-3 %. In the second part of the work, different patches were prepared and tested ex-vivo. Overall, the results obtained seem to highlight that drug loading, rather than the components of the adhesive matrix, plays the most relevant role for the permeation of trazodone. The addition of lauric acid, which produced a considerable enhancement in solution, was not effective when included in the patch. The obtained data are promising although probably not clinically relevant for the treatment of depression, but might be interesting for the treatment of insomnia and anxiety disorder, which require much lower doses., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

HIV-1 envelope glycoproteins (Env) from primary HIV-1 isolates typically adopt a pretriggered "closed" conformation that resists to CD4-induced (CD4i) non-neutralizing antibodies (nnAbs) mediating antibody-dependent cellular cytotoxicity (ADCC). CD4-mimetic compounds (CD4mcs) "open-up" Env allowing binding of CD4i nnAbs, thereby sensitizing HIV-1-infected cells to ADCC. Two families of CD4i nnAbs, the anti-cluster A and anti-coreceptor binding site (CoRBS) Abs, are required to mediate ADCC in combination with the indane CD4mc BNM-III-170. Recently, new indoline CD4mcs with improved potency and breadth have been described. Here, we show that the lead indoline CD4mc, CJF-III-288, sensitizes HIV-1-infected cells to ADCC mediated by anti-CoRBS Abs alone, contributing to improved ADCC activity. Structural and conformational analyses reveal that CJF-III-288, in combination with anti-CoRBS Abs, potently stabilizes an asymmetric "open" State-3 Env conformation, This Env conformation orients the anti-CoRBS Ab to improve ADCC activity and therapeutic potential.

Natural Killer (NK) cells have the potential to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC). NK cell activation is tightly regulated by the engagement of its inhibitory and activating receptors. The activating receptor CD16 drives ADCC upon binding to the Fc portion of antibodies; NK cell activation is further sustained by the co-engagement of activating receptors NTB-A and 2B4. During HIV-1 infection, Nef and Vpu accessory proteins contribute to ADCC escape by downregulating the ligands of NTB-A and 2B4. HIV-1 also evades ADCC by keeping its envelope glycoproteins (Env) in a "closed" conformation which effectively masks epitopes recognized by non-neutralizing antibodies (nnAbs) which are abundant in the plasma of people living with HIV. To achieve this, the virus uses its accessory proteins Nef and Vpu to downregulate the CD4 receptor, which otherwise interacts with Env and exposes the epitopes recognized by nnAbs. Small CD4-mimetic compounds (CD4mc) have the capacity to expose these epitopes, thus sensitizing infected cells to ADCC. Given the central role of NK cell co-activating receptors NTB-A and 2B4 in Fc-effector functions, we studied their contribution to CD4mc-mediated ADCC. Despite the fact that their ligands are partially downregulated by HIV-1, we found that both co-activating receptors significantly contribute to CD4mc sensitization of HIV-1-infected cells to ADCC.

CD4-mimetics (CD4mcs) are small molecule compounds that mimic the interaction of the CD4 receptor with HIV-1 envelope glycoproteins (Env). Env from primary viruses normally samples a "closed" conformation which occludes epitopes recognized by CD4-induced (CD4i) non-neutralizing antibodies (nnAbs). CD4mcs induce conformational changes on Env resulting in the exposure of these otherwise inaccessible epitopes. Here we evaluated the capacity of plasma from a cohort of 50 people living with HIV to recognize HIV-1-infected cells and eliminate them by antibody-dependent cellular cytotoxicity (ADCC) in the presence of a potent indoline CD4mc. We observed a marked heterogeneity among plasma samples. By measuring the levels of different families of CD4i Abs, we found that the levels of anti-cluster A, anti-coreceptor binding site and anti-gp41 cluster I antibodies are responsible for plasma-mediated ADCC in presence of CD4mc.

Plasma RNAemia, delayed antibody responses and inflammation predict COVID-19 outcomes, but the mechanisms underlying these immunovirological patterns are poorly understood. We profile 782 longitudinal plasma samples from 318 hospitalized patients with COVID-19. Integrated analysis using k-means reveals four patient clusters in a discovery cohort: mechanically ventilated critically-ill cases are subdivided into good prognosis and high-fatality clusters (reproduced in a validation cohort), while non-critical survivors segregate into high and low early antibody responders. Only the high-fatality cluster is enriched for transcriptomic signatures associated with COVID-19 severity, and each cluster has distinct RBD-specific antibody elicitation kinetics. Both critical and non-critical clusters with delayed antibody responses exhibit sustained IFN signatures, which negatively correlate with contemporaneous RBD-specific IgG levels and absolute SARS-CoV-2-specific B and CD4 + T cell frequencies. These data suggest that the "Interferon paradox" previously described in murine LCMV models is operative in COVID-19, with excessive IFN signaling delaying development of adaptive virus-specific immunity., (© 2024. The Author(s).)

Background: Nicotinamide adenine dinucleotide phosphate oxidase complex 2 (NOX2) deficiency, or chronic granulomatous disease (CGD), is an inborn error of immunity associated with increased susceptibility to infection and inflammatory manifestations. The pathophysiologic mechanism leading to the increased inflammatory response in CGD remains elusive., Objective: We investigated the pathophysiologic mechanisms leading to NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation in NOX2 deficiency., Methods: We used NOX2-deficient human primary and CRISPR-engineered macrophages to show that NOX2 deficiency enhances the inflammatory response mainly by modulating the 2 steps of NLRP3 inflammasome activation: its transcriptional priming and its posttranslational triggering., Results: At the transcriptional level, NOX2-deficient phagocytes display increased priming of the NLRP3 inflammasome, as evidenced by increased transcription of NLRP3 and IL-1β through an IL-1β-dependent stimulation of the nuclear factor kappa-light-chain enhancer of activated B cells (aka NF-κB) pathway. At the posttranslational level, the absence of NOX2 triggers the NLRP3 inflammasome activation by increased K + efflux and excessive release of mitochondrial DNA due to mitochondrial damage. Furthermore, NLRP3-driven pyroptosis in NOX2-deficient phagocytes further enhances NLRP3 activation by increasing K + efflux., Conclusion: Our results unveil the role of NOX2 as a repressor of the inflammatory response at both transcriptional and posttranslational levels and pave the way for a more targeted approach to treating CGD patients with inflammatory manifestations., (Copyright © 2023 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

While mRNA SARS-CoV-2 vaccination elicits strong humoral responses in the general population, humoral responses in people living with HIV (PLWH) remain to be clarified. Here, we conducted a longitudinal study of vaccine immunogenicity elicited after two and three doses of mRNA SARS-CoV-2 vaccine in PLWH stratified by their CD4 count. We measured the capacity of the antibodies elicited by vaccination to bind the Spike glycoprotein of different variants of concern (VOCs). We also evaluated the Fc-mediated effector functions of these antibodies by measuring their ability to eliminate CEM.NKr cells stably expressing SARS-CoV-2 Spikes. Finally, we measured the relative capacity of the antibodies to neutralize authentic SARS-CoV-2 virus after the third dose of mRNA vaccine. We found that after two doses of SARS-CoV-2 mRNA vaccine, PLWH with a CD4 count < 250/mm 3 had lower levels of anti-RBD IgG antibodies compared to PLWH with a CD4 count > 250/mm 3 ( p < 0.05). A third dose increased these levels and importantly, no major differences were observed in their capacity to mediate Fc-effector functions and neutralize authentic SARS-CoV-2. Overall, our work demonstrates the importance of mRNA vaccine boosting in immuno-compromised individuals presenting low levels of CD4.

HIV-1 evades antibody-dependent cellular cytotoxicity (ADCC) responses not only by controlling Env conformation and quantity at the cell surface but also by altering NK cell activation via the downmodulation of several ligands of activating and co-activating NK cell receptors. The signaling lymphocyte activation molecule (SLAM) family of receptors, which includes NTB-A and 2B4, act as co-activating receptors to sustain NK cell activation and cytotoxic responses. These receptors cooperate with CD16 (FcγRIII) and other activating receptors to trigger NK cell effector functions. In that context, Vpu-mediated downregulation of NTB-A on HIV-1-infected CD4 T cells was shown to prevent NK cell degranulation via an homophilic interaction, thus contributing to ADCC evasion. However, less is known on the capacity of HIV-1 to evade 2B4-mediated NK cell activation and ADCC. Here, we show that HIV-1 downregulates the ligand of 2B4, CD48, from the surface of infected cells in a Vpu-dependent manner. This activity is conserved among Vpu proteins from the HIV-1/SIVcpz lineage and depends on conserved residues located in its transmembrane domain and dual phosphoserine motif. We show that NTB-A and 2B4 stimulate CD16-mediated NK cell degranulation and contribute to ADCC responses directed to HIV-1-infected cells to the same extent. Our results suggest that HIV-1 has evolved to downmodulate the ligands of both SLAM receptors to evade ADCC. IMPORTANCE Antibody-dependent cellular cytotoxicity (ADCC) can contribute to the elimination of HIV-1-infected cells and HIV-1 reservoirs. An in-depth understanding of the mechanisms used by HIV-1 to evade ADCC might help develop novel approaches to reduce the viral reservoirs. Members of the signaling lymphocyte activation molecule (SLAM) family of receptors, such as NTB-A and 2B4, play a key role in stimulating NK cell effector functions, including ADCC. Here, we show that Vpu downmodulates CD48, the ligand of 2B4, and this contributes to protect HIV-1-infected cells from ADCC. Our results highlight the importance of the virus to prevent the triggering of the SLAM receptors to evade ADCC., Competing Interests: The authors declare no conflict of interest.

The ability of the HIV-1 accessory proteins Nef and Vpu to decrease CD4 levels contributes to the protection of infected cells from antibody-dependent cellular cytotoxicity (ADCC) by preventing the exposure of Env vulnerable epitopes. Small-molecule CD4 mimetics (CD4mc) based on the indane and piperidine scaffolds such as (+)-BNM-III-170 and ( S )-MCG-IV-210 sensitize HIV-1-infected cells to ADCC by exposing CD4-induced (CD4i) epitopes recognized by non-neutralizing antibodies that are abundantly present in plasma from people living with HIV. Here, we characterize a new family of CD4mc, ( S )-MCG-IV-210 derivatives, based on the piperidine scaffold which engages the gp120 within the Phe43 cavity by targeting the highly conserved Asp 368 Env residue. We utilized structure-based approaches and developed a series of piperidine analogs with improved activity to inhibit the infection of difficult-to-neutralize tier-2 viruses and sensitize infected cells to ADCC mediated by HIV+ plasma. Moreover, the new analogs formed an H-bond with the α-carboxylic acid group of Asp 368 , opening a new avenue to enlarge the breadth of this family of anti-Env small molecules. Overall, the new structural and biological attributes of these molecules make them good candidates for strategies aimed at the elimination of HIV-1-infected cells.

Human natural history and vaccine studies support a protective role of antibody dependent cellular cytotoxicity (ADCC) activity against many infectious diseases. One setting where this has consistently been observed is in HIV-1 vertical transmission, where passively acquired ADCC activity in HIV-exposed infants has correlated with reduced acquisition risk and reduced pathogenesis in HIV+ infants. However, the characteristics of HIV-specific antibodies comprising a maternal plasma ADCC response are not well understood. Here, we reconstructed monoclonal antibodies (mAbs) from memory B cells from late pregnancy in mother MG540, who did not transmit HIV to her infant despite several high-risk factors. Twenty mAbs representing 14 clonal families were reconstructed, which mediated ADCC and recognized multiple HIV Envelope epitopes. In experiments using Fc-defective variants, only combinations of several mAbs accounted for the majority of plasma ADCC of MG540 and her infant. We present these mAbs as evidence of a polyclonal repertoire with potent HIV-directed ADCC activity., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Cellular immune defects associated with suboptimal responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccination in people receiving hemodialysis (HD) are poorly understood. We longitudinally analyze antibody, B cell, CD4 + , and CD8 + T cell vaccine responses in 27 HD patients and 26 low-risk control individuals (CIs). The first two doses elicit weaker B cell and CD8 + T cell responses in HD than in CI, while CD4 + T cell responses are quantitatively similar. In HD, a third dose robustly boosts B cell responses, leads to convergent CD8 + T cell responses, and enhances comparatively more T helper (T H ) immunity. Unsupervised clustering of single-cell features reveals phenotypic and functional shifts over time and between cohorts. The third dose attenuates some features of T H cells in HD (tumor necrosis factor alpha [TNFα]/interleukin [IL]-2 skewing), while others (CCR6, CXCR6, programmed cell death protein 1 [PD-1], and HLA-DR overexpression) persist. Therefore, a third vaccine dose is critical to achieving robust multifaceted immunity in hemodialysis patients, although some distinct T H characteristics endure., Competing Interests: Declaration of interests C.T. serves as a consultant for Merck, Gilead, GSK, AstraZeneca, and Medicago., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Fenretinide (4-HPR), a retinoid derivative, has shown high antitumor activity, a low toxicological profile, and no induction of resistance. Despite these favorable features, the variability in oral absorption due to its low solubility combined with the high hepatic first pass effect strongly reduce clinical outcomes. To overcome the solubility and dissolution challenges of poorly water-soluble 4-HPR, we prepared a solid dispersion of the drug (4-HPR-P5) using a hydrophilic copolymer (P5) previously synthesized by our team as the solubilizing agent. The molecularly dispersed drug was obtained by antisolvent co-precipitation, an easy and up-scalable technique. A higher drug apparent solubility (1134-fold increase) and a markedly faster dissolution were obtained. In water, the colloidal dispersion showed a mean hydrodynamic diameter of 249 nm and positive zeta potential (+41.3 mV), confirming the suitability of the formulation for intravenous administration. The solid nanoparticles were also characterized by a high drug payload (37%), as was also evidenced by a chemometric-assisted Fourier transform infrared spectroscopy (FTIR) investigation. The 4-HPR-P5 exhibited antiproliferative activity, with IC50 values of 1.25 and 1.93 µM on IMR-32 and SH-SY5Y neuroblastoma cells, respectively. Our data confirmed that the 4-HPR-P5 formulation developed herein was able to increase drug apparent aqueous solubility and provide an extended release over time, thus suggesting that it represents an efficient approach to improve 4-HPR bioavailability., Competing Interests: The authors declare no conflict of interest.

HIV-1 envelope (Env) conformation determines the susceptibility of infected CD4 + T cells to antibody-dependent cellular cytotoxicity (ADCC). Upon interaction with CD4, Env adopts more "open" conformations, exposing ADCC epitopes. HIV-1 limits Env-CD4 interaction and protects infected cells against ADCC by downregulating CD4 via Nef, Vpu, and Env. Limited data exist, however, of the role of these proteins in downmodulating CD4 on infected macrophages and how this impacts Env conformation. While Nef, Vpu, and Env are all required to efficiently downregulate CD4 on infected CD4 + T cells, we show here that any one of these proteins is sufficient to downmodulate most CD4 from the surface of infected macrophages. Consistent with this finding, Nef and Vpu have a lesser impact on Env conformation and ADCC sensitivity in infected macrophages compared with CD4 + T cells. However, treatment of infected macrophages with small CD4 mimetics exposes vulnerable CD4-induced Env epitopes and sensitizes them to ADCC., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Due to the recrudescence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections worldwide, mainly caused by the Omicron variant of concern (VOC) and its sub-lineages, several jurisdictions are administering an mRNA vaccine boost. Here, we analyze humoral responses induced after the second and third doses of an mRNA vaccine in naive and previously infected donors who received their second dose with an extended 16-week interval. We observe that the extended interval elicits robust humoral responses against VOCs, but this response is significantly diminished 4 months after the second dose. Administering a boost to these individuals brings back the humoral responses to the same levels obtained after the extended second dose. Interestingly, we observe that administering a boost to individuals that initially received a short 3- to 4-week regimen elicits humoral responses similar to those observed in the long interval regimen. Nevertheless, humoral responses elicited by the boost in naive individuals do not reach those present in previously infected vaccinated individuals., Competing Interests: Declaration of interests A.R.G. is a consultant for Relation Therapeutics. E.J.W. is consulting for or is an advisor for Merck, Marengo, Janssen, Related Sciences, Synthekine, and Surface Oncology. E.J.W. is a founder of Surface Oncology, Danger Bio, and Arsenal Biosciences., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

SARS-CoV-2 continues to infect millions of people worldwide. The subvariants arising from the variant-of-concern (VOC) Omicron include BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4, and BA.5. All possess multiple mutations in their Spike glycoprotein, notably in its immunogenic receptor-binding domain (RBD), and present enhanced viral transmission. The highly mutated Spike glycoproteins from these subvariants present different degrees of resistance to recognition and cross-neutralisation by plasma from previously infected and/or vaccinated individuals. We have recently shown that the temperature affects the interaction between the Spike and its receptor, the angiotensin converting enzyme 2 (ACE2). The affinity of RBD for ACE2 is significantly increased at lower temperatures. However, whether this is also observed with the Spike of Omicron and sub-lineages is not known. Here we show that, similar to other variants, Spikes from Omicron sub-lineages bind better the ACE2 receptor at lower temperatures. Whether this translates into enhanced transmission during the fall and winter seasons remains to be determined.

Background: Plateletpheresis involves platelet separation and collection from whole blood while other blood cells are returned to the donor. Because platelets are replaced faster than red blood cells, as many as 24 donations can be done annually. However, some frequent apheresis platelet donors (>20 donations annually) display severe plateletpheresis-associated lymphopenia; in particular, CD4 + T but not B cell numbers are decreased. COVID-19 vaccination thereby provides a model to assess whether lymphopenic platelet donors present compromised humoral immune responses., Study Design and Methods: We assessed vaccine responses following 2 doses of COVID-19 vaccination in a cohort of 43 plateletpheresis donors with a range of pre-vaccination CD4 + T cell counts (76-1537 cells/μl). In addition to baseline T cell measurements, antibody binding assays to full-length Spike and the Receptor Binding Domain (RBD) were performed pre- and post-vaccination. Furthermore, pseudo-particle neutralization and antibody-dependent cellular cytotoxicity assays were conducted to measure antibody functionality., Results: Participants were stratified into two groups: <400 CD4/μl (n = 27) and ≥ 400 CD4/μl (n = 16). Following the first dose, 79% seroconverted within the <400 CD4/μl group compared to 87% in the ≥400 CD4/μl group; all donors were seropositive post-second dose with significant increases in antibody levels. Importantly differences in CD4 + T cell levels minimally impacted neutralization, Spike recognition, and IgG Fc-mediated effector functions., Discussion: Overall, our results indicate that lymphopenic plateletpheresis donors do not exhibit significant immune dysfunction; they have retained the T and B cell functionality necessary for potent antibody responses after vaccination., (© 2022 AABB.)

Usually, the administration of drugs by feeding tube in dysphagic patients involves handling of marketing licenses outside their term, due to the lack of suitable formulations. This circumstance has put health professionals in the dilemma of choosing the formulation whose manipulation possibly does not alter the effectiveness of the drug. In this regard, a practical guide providing indications on the prescription, handling, and administration of drugs through enteral feeding tube could be of paramount utility. For this purpose, we have considered the 1047 solid oral pharmaceutical forms included in the formulary of San Paolo Hospital (Savona, Italy). From our analysis, it emerges that 95% of medicinal products are worryingly used off-label and 40% have to be managed by the hospital pharmacists without having suitable indications by either the manufacturers or by literature studies. To fill this gap, we have compiled a detailed table containing missing indications derived from pharmacist expertise and evidence-based practices, with the aim that the sharing of our procedures will contribute to make uniform pharmacological therapies from one hospital to another. This study will allow doctors to have easy access to information on drugs that can be prescribed and nurses to become familiar only with the pharmaceutical forms that can be administered.

Patients receiving hemodialysis (HD) have more inflammatory monocytes and less plasmacytoid dendritic cells (DCs) compared with healthy controls.Patients on HD who have a poor antibody response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine had fewer monocyte-derived DCs and conventional DCs compared with good responders.The defects in antigen presentation might be possible therapeutic targets to increase vaccine efficacy in HD patients., Competing Interests: A. Finzi reports receiving research funding and honoraria from, and serving in an advisory or leadership role for, ViiV. C. Lamarche reports having the patents PCT/CA2018/051167 and PCT/CA2018/051174 filed. R.S. Suri reports receiving honoraria from Amgen and Otskuka; and serving in an advisory or leadership role for Canadian Institutes of Health Research Institute of Circulatory and Respiratory Health and Canadian Society of Nephrology. All remaining authors have nothing to disclose., (Copyright © 2022 by the American Society of Nephrology.)

Soluble angiotensin-converting enzyme 2 (ACE2) constitutes an attractive antiviral capable of targeting a wide range of coronaviruses using ACE2 as their receptor. Using structure-guided approaches, we developed a series of bivalent ACE2-Fcs harboring functionally and structurally validated mutations that enhance severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain recognition by up to ~12-fold and remove angiotensin enzymatic activity. The lead variant M81 potently cross-neutralized SARS-CoV-2 variants of concern (VOCs), including Omicron, at subnanomolar half-maximal inhibitory concentration and was capable of robust Fc-effector functions, including antibody-dependent cellular cytotoxicity, phagocytosis, and complement deposition. When tested in a stringent K18-hACE2 mouse model, Fc-enhanced ACE2-Fc delayed death by 3 to 5 days or effectively resolved lethal SARS-CoV-2 infection in both prophylactic and therapeutic settings via the combined effects of neutralization and Fc-effector functions. These data add to the demonstrated utility of soluble ACE2 as a valuable SARS-CoV-2 antiviral and indicate that Fc-effector functions may constitute an important component of ACE2-Fc therapeutic activity.

Spacing of BNT162b2 mRNA doses beyond 3 weeks raises concerns about vaccine efficacy. We longitudinally analyze B cell, T cell, and humoral responses to two BNT162b2 mRNA doses administered 16 weeks apart in 53 SARS-CoV-2 naive and previously infected donors. This regimen elicits robust RBD-specific B cell responses whose kinetics differs between cohorts, the second dose leading to increased magnitude in naive participants only. While boosting does not increase magnitude of CD4 + T cell responses further compared with the first dose, unsupervised clustering of single-cell features reveals phenotypic and functional shifts over time and between cohorts. Integrated analysis shows longitudinal immune component-specific associations, with early T helper responses post first dose correlating with B cell responses after the second dose, and memory T helper generated between doses correlating with CD8 T cell responses after boosting. Therefore, boosting elicits a robust cellular recall response after the 16-week interval, indicating functional immune memory., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The heavily glycosylated HIV-1 envelope glycoprotein (Env) is the sole viral antigen present at the surface of virions and infected cells, representing the main target for antibody responses. The FDA-approved small molecule temsavir acts as an HIV-1 attachment inhibitor by preventing Env-CD4 interaction. This molecule also stabilizes Env in a prefusion "closed" conformation that is preferentially targeted by several broadly neutralizing antibodies (bNAbs). A recent study showed that an analog of temsavir (BMS-377806) affects the cleavage and addition of complex glycans on Env. In this study, we investigated the impact of temsavir on the overall glycosylation, proteolytic cleavage, cell surface expression, and antigenicity of Env. We found that temsavir impacts Env glycosylation and processing at physiological concentrations. This significantly alters the capacity of several bNAbs to recognize Env present on virions and HIV-1-infected cells. Temsavir treatment also reduces the capacity of bNAbs to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC). Consequently, the impact of temsavir on Env glycosylation and antigenicity should be considered for the development of new antibody-based approaches in temsavir-treated individuals. IMPORTANCE FDA-approved fostemsavir, the prodrug for the active moiety small molecule temsavir (GSK 2616713 [formally BMS-626529]), acts as an attachment inhibitor by targeting the HIV-1 envelope (Env) and preventing CD4 interaction. Temsavir also stabilizes Env in its "closed," functional state 1 conformation, which represents an ideal target for broadly neutralizing antibodies (bNAbs). Since these antibodies recognize conformation-dependent epitopes composed of or adjacent to glycans, we evaluated the impact of temsavir treatment on overall Env glycosylation and its influence on bNAb recognition. Our results showed an alteration of Env glycosylation and cleavage by temsavir at physiological concentrations. This significantly modifies the overall antigenicity of Env and therefore reduces the capacity of bNAbs to recognize and eliminate HIV-1-infected cells by ADCC. These findings provide important information for the design of immunotherapies aimed at targeting the viral reservoir in temsavir-treated individuals.

Introduction: COVID-19 vaccine efficacy has been evaluated in large clinical trials and in real-world situation. Although they have proven to be very effective in the general population, little is known about their efficacy in immunocompromised patients. HIV-infected individuals' response to vaccine may vary according to the type of vaccine and their level of immunosuppression. We evaluated immunogenicity of an mRNA anti-SARS CoV-2 vaccine in HIV-positive individuals., Methods: HIV-positive individuals (n = 121) were recruited from HIV clinics in Montreal and stratified according to their CD4 counts. A control group of 20 health care workers naïve to SARS CoV-2 was used. The participants' Anti-RBD IgG responses were measured by ELISA at baseline and 3-4 weeks after receiving the first dose of an mRNA vaccine)., Results: Eleven of 121 participants had anti-COVID-19 antibodies at baseline, and a further 4 had incomplete data for the analysis. Mean anti-RBD IgG responses were similar between the HIV negative control group (n = 20) and the combined HIV+ group (n = 106) (p = 0.72). However, these responses were significantly lower in the group with <250 CD4 cells/mm 3 . (p < 0.0001). Increasing age was independently associated with decreased immunogenicity., Conclusion: HIV-positive individuals with CD4 counts over 250 cells/mm 3 have an anti-RBD IgG response similar to the general population. However, HIV-positive individuals with the lowest CD4 counts (<250 cells/mm 3 ) have a weaker response. These data would support the hypothesis that a booster dose might be needed in this subgroup of HIV-positive individuals, depending on their response to the second dose., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Continuous emergence of SARS-CoV-2 variants of concern (VOCs) is fueling the COVID-19 pandemic. Omicron (B.1.1.529) rapidly spread worldwide. The large number of mutations in its Spike raise concerns about a major antigenic drift that could significantly decrease vaccine efficacy and infection-induced immunity. A long interval between BNT162b2 mRNA doses elicits antibodies that efficiently recognize Spikes from different VOCs. Here, we evaluate the recognition of Omicron Spike by plasma from a cohort of SARS-CoV-2 naive and previously infected individuals who received their BNT162b2 mRNA vaccine 16 weeks apart. Omicron Spike is recognized less efficiently than D614G, Alpha, Beta, Gamma, and Delta Spikes. We compare with plasma activity from participants receiving a short (4 weeks) interval regimen. Plasma from individuals of the long-interval cohort recognize and neutralize better the Omicron Spike compared with those who received a short interval. Whether this difference confers any clinical benefit against Omicron remains unknown., Competing Interests: Declaration of interests A.F. has filed a provisional patent application on the anti-Spike monoclonal antibody CV3-25., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the treatment of pediatric diseases, mass-produced dosage forms are often not suitable for children. Commercially available medicines are commonly manipulated and mixed with food by caregivers at home, or extemporaneous medications are routinely compounded in the hospital pharmacies to treat hospitalized children. Despite considerable efforts by regulatory agencies, the pediatric population is still exposed to questionable and potentially harmful practices. When designing medicines for children, the ability to fine-tune the dosage while ensuring the safety of the ingredients is of paramount importance. For these purposes solid formulations may represent a valid alternative to liquid formulations for their simpler formula and more stability, and, to overcome the problem of swelling ability, mini-tablets could be a practicable option. This review deals with the different approaches that may be applied to develop mini-tablets intended for pediatrics with a focus on the safety of excipients. Alongside the conventional method of compression, 3D printing appeared particularly appealing, as it allows to reduce the number of ingredients and to avoid both the mixing of powders and intermediate steps such as granulation. Therefore, this technique could be well adaptable to the daily galenic preparations of a hospital pharmacy, thus leading to a reduction of the common practice of off-label preparations.

The standard regimen of the BNT162b2 mRNA vaccine for SARS-CoV-2 includes two doses administered three weeks apart. However, some public health authorities spaced these doses, raising questions about efficacy. We analyzed longitudinal humoral responses against the D614G strain and variants of concern for SARS-CoV-2 in a cohort of SARS-CoV-2-naive and previously infected individuals who received the BNT162b2 mRNA vaccine with sixteen weeks between doses. While administering a second dose to previously infected individuals did not significantly improve humoral responses, these responses significantly increased in naive individuals after a 16-week spaced second dose, achieving similar levels as in previously infected individuals. Comparing these responses to those elicited in individuals receiving a short (4-week) dose interval showed that a 16-week interval induced more robust responses among naive vaccinees. These findings suggest that a longer interval between vaccine doses does not compromise efficacy and may allow greater flexibility in vaccine administration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Children affected by chronic liver disease exhibit impaired neurocognitive development and growth due to the low absorption and digestion of nutrients. Furthermore, malnutrition is an adverse prognostic factor in liver transplantation as it is associated with an increase in morbidity and mortality. D-α-tocopheryl-polyethylene-glycol-succinate (TPGS) is currently administered per os as a vitamin E source to improve children's survival and well-being; however, TPGS alone does not reverse spinocerebellar degeneration and lipid peroxidation. To potentiate the effects of TPGS, we loaded micelles with resveratrol (RES), a natural polyphenol, with antioxidant and antiinflammatory activities, which has demonstrated protective action in the liver. Firstly, we investigated the suitability of TPGS to encapsulate RES in micelles by means of a phase-solubility study, then RES-TPGS formulations were prepared via solvent casting and solvent diffusion evaporation methods. RES-TPGS colloidal dispersions showed small mean diameters (12 nm), low polydispersity, and quite neutral Zeta potentials. The formulations showed a sustained drug release and a good drug loading capacity, further confirmed by infrared spectroscopy and differential scanning calorimetry. RES-TPGSs exhibited unaltered antioxidant activity compared to pristine RES via the DPPH assay and a significant reduction in toxicity compared to empty TPGS on HaCaT cells. Thus, RES-TPGS micelles may overcome the challenges of current liver disease therapy by providing more protective effects thanks to the antioxidant activity of RES and by reducing the surfactant toxicity on normal cells.

All- trans -retinoic acid (ATRA) represents the first-choice treatment for several skin diseases, including epithelial skin cancer and acne. However, ATRA's cutaneous side effects, like redness and peeling, and its high instability limit its efficacy. To address these drawbacks and to improve ATRA solubilization, we prepared ATRA-loaded micelles (ATRA-TPGSs), by its encapsulation in D-α-tocopheryl-polyethylene-glycol-succinate (TPGS). First, to explore the feasibility of the project, a solubility study based on the equilibrium method was performed; then, six ATRA-TPGS formulations were prepared by the solvent-casting method using different TPGS amounts. ATRA-TPGSs showed small sizes (11-20 nm), low polydispersity, slightly negative zeta potential, and proved good encapsulation efficiency, confirmed by a chemometric-assisted Fourier transform infrared spectroscopy (FTIR) investigation. ATRA-TPGS stability was also investigated to choose the most stable formulation. Using Carbopol ® 980 as gelling agent, ATRA-TPGS-loaded gels were obtained and analyzed for their rheological profiles. Ex vivo release studies from ATRA-TPGSs were performed by Franz cells, demonstrating a permeation after 24 h of 22 ± 4 µ cm -2 . ATRA-TPGSs showed enhanced cytotoxic effects on melanoma cells, suggesting that these formulations may represent a valid alternative to improve patient compliance and to achieve more efficacious therapeutic outcomes.

Gain of function (GOF) mutations affecting the inflammasome component NLRC4 are known to cause early-onset macrophage activation syndrome (MAS) and neonatal enterocolitis. Here we report a patient with a NLRC4 GOF mutation presenting with neonatal MAS efficiently treated with a combination of anakinra and rapamycin. Through in vitro studies, we show that rapamycin reduces both IL-1β and IL-18 secretion by the patient's phagocytic cells. The reduction of cytokine secretion is associated with a reduction of caspase-1 activation regardless of the pathogen- or danger-associated molecular patterns triggering the activation of the inflammasome. This study suggests that patients with inherited auto-inflammatory disorders could benefit from an adjunctive therapy with rapamycin.

The effectiveness of the new powder mixer Canguro J tumbler was evaluated using lactose, microcrystalline cellulose, and salicylic acid as chemical indicator with the ratio 88:10:2 (w/w). The mixing time, the speed of the tumbler (rpm), its inclination, and filling percentage were varied in order to assess the limits of the mixer and the best parameters to use for obtaining a mixture as uniform as possible. The same experiments were then repeated after addition of 1% (w/w) magnesium stearate to the mixture of powders. The efficiency in the distribution of this lubricant was estimated by the progressive hardness reduction of the tablets derived from the compression of the powders, at a constant applied force. Finally, a comparison between Canguro J and a very efficient V-shaped mixer of the same capacity was performed. The results show that all investigated parameters influenced the mixing capability of Canguro J. The best effectiveness of the mixer occurred at the filling rate of 50% and a rotation speed of 20 rpm; in this case, Canguro J is even a little more effective than the V-shaped mixer. However, even at the filling rate of 70%, the same distribution uniformity of the powders can be obtained after a mixing time protraction of a few minutes.