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PURPOSE: The purpose of this study was to determine the role of transcutaneous retrobulbar amphotericin B (TRAMB) injection in COVID-19-related rhino-orbital-cerebral mucormycosis (ROCM). MATERIALS AND METHODS: This prospective interventional case series included 7 eyes of 7 patients diagnosed with COVID-19 ROCM presenting from May to September 2021 at a tertiary care center in North India. Active or recovered cases of COVID-19 with clinical features suggestive of ROCM and early orbital involvement were included. Following functional endoscopic sinus surgery, patients were given TRAMB 3.5 mg/ml in single ocular quadrant depending upon the muscle involved on contrast-enhanced magnetic resonance imaging (MRI). Patients were followed up for a period of 12 weeks. RESULTS: All patients were diabetic and COVID-19 reverse transcription-polymerase chain reaction positive. The mean age of patients was 48.4 years ± 11 years, out of which 5 patients were male and 2 were female. The left eye was involved in 57.14% of cases whereas 42.85% of cases had right eye involvement. Presenting complaints were drooping of the upper lid (57.14%; n = 4), proptosis (28.57%; n = 2), facial pain and swelling (85.71%; n = 6), and nasal block (57.14%; n = 4). Stagewise distribution was 3a in four patients (57.14%), 3b in two patients (28.57%), and 4a in one patient. TRAMB injections (median - 3) were given, and improvement was seen in all patients in terms of ocular movements, ptosis, proptosis, and muscle enhancement on MRI scan. Three patients had an increase in intraocular pressure and inflammation which settled within a week with favorable anatomical and functional outcomes. CONCLUSION: Transcutaneous retrobulbar amphotericin B should be considered an adjunctive treatment modality for ROCM patients with limited orbital disease to preserve ocular function.
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Kidney transplant recipients are at an increased risk of hospitalisation and death from SARS-CoV-2 infection, and standard two-dose vaccination schedules are typically inadequate to generate protective immunity. Gut dysbiosis, which is common among kidney transplant recipients and known to effect systemic immunity, may be a contributing factor to a lack of vaccine immunogenicity in this at-risk cohort. The gut microbiota modulates vaccine responses, with the production of immunomodulatory short-chain fatty acids by bacteria such as Bifidobacterium associated with heightened vaccine responses in both observational and experimental studies. As SCFA-producing populations in the gut microbiota are enhanced by diets rich in non-digestible fibre, dietary supplementation with prebiotic fibre emerges as a potential adjuvant strategy to correct dysbiosis and improve vaccine-induced immunity. In a randomised, double-bind, placebo-controlled trial of 72 kidney transplant recipients, we found dietary supplementation with prebiotic inulin for 4 weeks before and after a third SARS-CoV2 mRNA vaccine to be feasible, tolerable, and safe. Inulin supplementation resulted in an increase in gut Bifidobacterium, as determined by 16S RNA sequencing, but did not increase in vitro neutralisation of live SARS-CoV-2 virus at 4 weeks following a third vaccination. Dietary fibre supplementation is a feasible strategy with the potential to enhance vaccine-induced immunity and warrants further investigation.
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In July/August 2023, the highly mutated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) BA.2.86 lineage emerged and its descendant JN.1 is on track to become the dominant SARS-CoV-2 lineage globally. Compared to the spike (S) protein of the parental BA.2.86 lineage, the JN.1 S protein contains one mutation, L455S, which may affect receptor binding and antibody evasion. Here, we performed a virological assessment of the JN.1 lineage employing pseudovirus particles bearing diverse SARS-CoV-2 S proteins. Using this strategy, it was found that S protein mutation L455S confers increased neutralization resistance but reduces ACE2 binding capacity and S protein-driven cell entry efficiency. Altogether, these data suggest that the benefit of increased antibody evasion outweighs the reduced ACE2 binding capacity and further enabled the JN.1 lineage to effectively spread in the human population.
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The development of novel antivirals is crucial not only for managing current COVID-19 infections but for addressing potential future zoonotic outbreaks. SARS-CoV-2 main protease (Mpro) is vital for viral replication and viability and therefore serves as an attractive target for antiviral intervention. Herein, we report the optimization of a cyclic peptide inhibitor that emerged from an mRNA display selection against the SARS-CoV-2 Mpro to enhance its cell permeability and inâ vitro antiviral activity. By identifying mutation-tolerant amino acid residues within the peptide sequence, we describe the development of a second-generation Mpro inhibitor bearing five cyclohexylalanine residues. This cyclic peptide analogue exhibited significantly improved cell permeability and antiviral activity compared to the parent peptide. This approach highlights the importance of optimizing cyclic peptide hits for activity against intracellular targets such as the SARS-CoV-2 Mpro.
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Antivirales , Proteasas 3C de Coronavirus , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos Cíclicos , SARS-CoV-2 , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Antivirales/química , Antivirales/farmacología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Proteasas 3C de Coronavirus/química , Humanos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Aminoácidos/química , Tratamiento Farmacológico de COVID-19RESUMEN
This study investigates the humoral and cellular immune responses and health-related quality of life measures in individuals with mild to moderate long COVID (LC) compared to age and gender matched recovered COVID-19 controls (MC) over 24 months. LC participants show elevated nucleocapsid IgG levels at 3 months, and higher neutralizing capacity up to 8 months post-infection. Increased spike-specific and nucleocapsid-specific CD4+ T cells, PD-1, and TIM-3 expression on CD4+ and CD8+ T cells were observed at 3 and 8 months, but these differences do not persist at 24 months. Some LC participants had detectable IFN-γ and IFN-ß, that was attributed to reinfection and antigen re-exposure. Single-cell RNA sequencing at the 24 month timepoint shows similar immune cell proportions and reconstitution of naïve T and B cell subsets in LC and MC. No significant differences in exhaustion scores or antigen-specific T cell clones are observed. These findings suggest resolution of immune activation in LC and return to comparable immune responses between LC and MC over time. Improvement in self-reported health-related quality of life at 24 months was also evident in the majority of LC (62%). PTX3, CRP levels and platelet count are associated with improvements in health-related quality of life.
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COVID-19 , Síndrome Post Agudo de COVID-19 , Humanos , Linfocitos T CD8-positivos , Calidad de Vida , SARS-CoV-2 , Anticuerpos AntiviralesRESUMEN
The development of effective antiviral compounds is essential for mitigating the effects of the COVID-19 pandemic. Entry of SARS-CoV-2 virions into host cells is mediated by the interaction between the viral spike (S) protein and membrane-bound angiotensin-converting enzyme 2 (ACE2) on the surface of epithelial cells. Inhibition of this viral protein-host protein interaction is an attractive avenue for the development of antiviral molecules with numerous spike-binding molecules generated to date. Herein, we describe an alternative approach to inhibit the spike-ACE2 interaction by targeting the spike-binding interface of human ACE2 via mRNA display. Two consecutive display selections were performed to direct cyclic peptide ligand binding toward the spike binding interface of ACE2. Through this process, potent cyclic peptide binders of human ACE2 (with affinities in the picomolar to nanomolar range) were identified, two of which neutralized SARS-CoV-2 entry. This work demonstrates the potential of targeting ACE2 for the generation of anti-SARS-CoV-2 therapeutics as well as broad spectrum antivirals for the treatment of SARS-like betacoronavirus infection.
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COVID-19 , SARS-CoV-2 , Humanos , Enzima Convertidora de Angiotensina 2/química , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/metabolismo , Pandemias , Ligandos , Unión Proteica , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Antivirales/farmacología , Antivirales/químicaRESUMEN
Graves' disease is a self-limiting autoimmune thyroid disorder caused by stimulating antibodies to the thyroid-stimulating hormone receptor. It usually affects middle-aged females in the fourth to sixth decade of life. It is distinguished by keratopathy, chemosis, proptosis, and eyelid swelling, in addition to ocular discomfort. A total of 3-5% of cases present with a severe form of Graves' orbitopathy, which manifests with diminution of vision, optic nerve compression, optic neuropathy, and exposure keratopathy. We describe a case of a 34-year-old female patient who presented with the chief complaint of rapid deterioration of vision over a period of three months in the right eye. Ocular examination revealed proptosis, widened palpebral aperture, elevation of intra-ocular pressure (IOP) in the upgaze, restricted eye movements, and signs of optic nerve compression. Findings were confirmed on a CT scan of the orbit. The unusual presentation in this case was that she had rapid, significant deterioration of vision in the right eye, with a progression of proptosis more marked in the contralateral eye. This underlies the importance of thoroughly examining for any possible orbital apex syndrome in both eyes, not just the eye with marked proptosis. The patient, being reluctant for orbital decompression, was prescribed IV methylprednisolone 1 g for three consecutive days, which reduced her proptosis and improved her vision. This acted as a temporary measure to increase the duration of the surgical window until the time the patient undergoes the surgery.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) breakthrough infection of vaccinated individuals is increasingly common with the circulation of highly immune evasive and transmissible Omicron variants. Here, we report the dynamics and durability of recalled spike-specific humoral immunity following Omicron BA.1 or BA.2 breakthrough infection, with longitudinal sampling up to 8 months after infection. Both BA.1 and BA.2 infections robustly boosted neutralization activity against the infecting strain while expanding breadth against BA.4, although neutralization activity was substantially reduced for the more recent XBB and BQ.1.1 strains. Cross-reactive memory B cells against both ancestral and Omicron spike were predominantly expanded by infection, with limited recruitment of de novo Omicron-specific B cells or antibodies. Modeling of neutralization titers predicts that protection from symptomatic reinfection against antigenically similar strains will be durable but is undermined by new emerging strains with further neutralization escape.
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Anticuerpos Neutralizantes , Infección Irruptiva , COVID-19 , Humanos , SARS-CoV-2RESUMEN
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a respiratory virus that causes COVID-19 disease, with an estimated global mortality of approximately 2%. While global response strategies, which are predominantly reliant on regular vaccinations, have shifted from zero COVID to living with COVID, there is a distinct lack of broad-spectrum direct acting antiviral therapies that maintain efficacy across evolving SARS-CoV-2 variants of concern. This is of most concern for immunocompromised and immunosuppressed individuals who lack robust immune responses following vaccination, and others at risk for severe COVID and long-COVID. RNA interference (RNAi) therapeutics induced by short interfering RNAs (siRNAs) offer a promising antiviral treatment option, with broad-spectrum antiviral capabilities unparalleled by current antiviral therapeutics and a high genetic barrier to antiviral escape. Here we describe novel siRNAs, targeting highly conserved regions of the SARS-CoV-1 and 2 genome of both human and animal species, with multi-variant antiviral potency against eight SARS-CoV-2 lineages - Ancestral VIC01, Alpha, Beta, Gamma, Delta, Zeta, Kappa and Omicron. Treatment with our siRNA resulted in significant protection against virus-mediated cell death in vitro, with >97% cell survival (P < 0.0001), and corresponding reductions of viral nucleocapsid RNA of up to 99.9% (P < 0.0001). When compared to antivirals; Sotrovimab and Remdesivir, the siRNAs demonstrated a more potent antiviral effect and similarly, when multiplexing siRNAs to target different viral regions simultaneously, an increased antiviral effect was observed compared to individual siRNA treatments (P < 0.0001). These results demonstrate the potential for a highly effective broad-spectrum direct acting antiviral against multiple SARS-CoV-2 variants, including variants resistant to antivirals and vaccine generated neutralizing antibodies.
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COVID-19 , Hepatitis C Crónica , Animales , Humanos , ARN Interferente Pequeño/genética , SARS-CoV-2/genética , Antivirales/farmacología , Antivirales/uso terapéutico , Síndrome Post Agudo de COVID-19 , COVID-19/terapia , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales , Glicoproteína de la Espiga del CoronavirusRESUMEN
Although a more efficient adaptive humoral immune response has been proposed to underlie the usually favorable outcome of pediatric COVID-19, the breadth of viral and vaccine cross-reactivity toward the ever-mutating Spike protein among variants of concern (VOCs) has not yet been compared between children and adults. We assessed antibodies to conformational Spike in COVID-19-naïve children and adults vaccinated by BNT162b2 and ChAdOx1, and naturally infected with SARS-CoV-2 Early Clade, Delta, and Omicron. Sera were analyzed against Spike including naturally occurring VOCs Alpha, Beta, Gamma, Delta, and Omicron BA.1, BA.2, BA.5, BQ.1.1, BA2.75.2, and XBB.1, and variants of interest Epsilon, Kappa, Eta, D.2, and artificial mutant Spikes. There was no notable difference between breadth and longevity of antibody against VOCs in children and adults. Vaccinated individuals displayed similar immunoreactivity profiles across variants compared with naturally infected individuals. Delta-infected patients had an enhanced cross-reactivity toward Delta and earlier VOCs compared to patients infected by Early Clade SARS-CoV-2. Although Omicron BA.1, BA.2, BA.5, BQ.1.1, BA2.75.2, and XBB.1 antibody titers were generated after Omicron infection, cross-reactive binding against Omicron subvariants was reduced across all infection, immunization, and age groups. Some mutations, such as 498R and 501Y, epistatically combined to enhance cross-reactive binding, but could not fully compensate for antibody-evasive mutations within the Omicron subvariants tested. Our results reveal important molecular features central to the generation of high antibody titers and broad immunoreactivity that should be considered in future vaccine design and global serosurveillance in the context of limited vaccine boosters available to the pediatric population.
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COVID-19 , Vacunas , Niño , Humanos , Adulto , SARS-CoV-2 , Formación de Anticuerpos , Vacuna BNT162 , AnticuerposRESUMEN
The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used messenger RNA (mRNA) display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor-binding domain, N-terminal domain, and S2 region, distal to the angiotensin-converting enzyme 2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Pandemias/prevención & control , Péptidos/farmacologíaRESUMEN
The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein subunit and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3-4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity next to Ala1016 and Ala1020 in the 3-4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. S glycoprotein membrane fusion function was retained with Ala1016/Ala1020 cavity-filling mutations associated with improved recombinant S2P-FHA thermostability, however 2 mutants, A1016L and A1016V/A1020I, lacked ability to mediate entry of S-HIV-1 pseudoparticles into 293-ACE2 cells. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited neutralizing antibody with 50%-inhibitory dilutions (ID50s) in the range 2,700-5,110 for ancestral and Delta-derived viruses, and 210-1,744 for Omicron BA.1. The antigens elicited antibody specificities directed to the receptor-binding domain (RBD), N-terminal domain (NTD), fusion peptide and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S2P-FHA-like ectodomain oligomers in the absence of an external trimerization motif (T4 foldon), thus representing an alternative approach for stabilizing oligomeric S glycoprotein vaccines.
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COVID-19 , Síndrome Respiratorio Agudo Grave , Humanos , Glicoproteína de la Espiga del Coronavirus , Enzima Convertidora de Angiotensina 2 , Anticuerpos NeutralizantesRESUMEN
BACKGROUND: The Omicron era of the COVID-19 pandemic commenced at the beginning of 2022 and whilst it started with primarily BA.1, it was latter dominated by BA.2 and the related sub-lineage BA.5. Following resolution of the global BA.5 wave, a diverse grouping of Omicron sub-lineages emerged derived from BA.2, BA.5 and recombinants thereof. Whilst emerging from distinct lineages, all shared similar changes in the Spike glycoprotein affording them an outgrowth advantage through evasion of neutralising antibodies. METHODS: Over the course of 2022, we monitored the potency and breadth of antibody neutralization responses to many emerging variants in the Australian community at three levels: (i) we tracked over 420,000 U.S. plasma donors over time through various vaccine booster roll outs and Omicron waves using sequentially collected IgG pools; (ii) we mapped the antibody response in individuals using blood from stringently curated vaccine and convalescent cohorts. (iii) finally we determine the in vitro efficacy of clinically approved therapies Evusheld and Sotrovimab. FINDINGS: In pooled IgG samples, we observed the maturation of neutralization breadth to Omicron variants over time through continuing vaccine and infection waves. Importantly, in many cases, we observed increased antibody breadth to variants that were yet to be in circulation. Determination of viral neutralization at the cohort level supported equivalent coverage across prior and emerging variants with isolates BQ.1.1, XBB.1, BR.2.1 and XBF the most evasive. Further, these emerging variants were resistant to Evusheld, whilst increasing neutralization resistance to Sotrovimab was restricted to BQ.1.1 and XBF. We conclude at this current point in time that dominant variants can evade antibodies at levels equivalent to their most evasive lineage counterparts but sustain an entry phenotype that continues to promote an additional outgrowth advantage. In Australia, BR.2.1 and XBF share this phenotype and, in contrast to global variants, are uniquely dominant in this region in the later months of 2022. INTERPRETATION: Whilst the appearance of a diverse range of omicron lineages has led to primary or partial resistance to clinically approved monoclonal antibodies, the maturation of the antibody response across both cohorts and a large donor pools importantly observes increasing breadth in the antibody neutralisation responses over time with a trajectory that covers both current and known emerging variants. FUNDING: This work was primarily supported by Australian Medical Foundation research grants MRF2005760 (SGT, GM & WDR), Medical Research Future Fund Antiviral Development Call grant (WDR), the New South Wales Health COVID-19 Research Grants Round 2 (SGT & FB) and the NSW Vaccine Infection and Immunology Collaborative (VIIM) (ALC). Variant modeling was supported by funding from SciLifeLab's Pandemic Laboratory Preparedness program to B.M. (VC-2022-0028) and by the European Union's Horizon 2020 research and innovation programme under grant agreement no. 101003653 (CoroNAb) to B.M.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Pandemias/prevención & control , COVID-19/prevención & control , Australia/epidemiología , Anticuerpos Neutralizantes , Inmunoglobulina G , Anticuerpos AntiviralesRESUMEN
BACKGROUND: Predominantly antibody deficiency (PAD) is the most common category of inborn errors of immunity and is underpinned by impaired generation of appropriate antibody diversity and quantity. In the clinic, responses are interrogated by assessment of vaccination responses, which is central to many PAD diagnoses. However, the composition of the generated antibody repertoire is concealed from traditional quantitative measures of serological responses. Leveraging modern mass spectrometry-based proteomics (MS-proteomics), it is possible to elaborate the molecular features of specific antibody repertoires, which may address current limitations of diagnostic vaccinology. OBJECTIVES: We sought to evaluate serum antibody responses in patients with PAD following vaccination with a neo-antigen (severe acute respiratory syndrome coronavirus-2 vaccination) using MS-proteomics. METHODS: Following severe acute respiratory syndrome coronavirus-2 vaccination, serological responses in individuals with PAD and healthy controls (HCs) were assessed by anti-S1 subunit ELISA and neutralization assays. Purified anti-S1 subunit IgG and IgM was profiled by MS-proteomics for IGHV subfamily usage and somatic hypermutation analysis. RESULTS: Twelve patients with PAD who were vaccine-responsive were recruited with 11 matched vaccinated HCs. Neutralization and end point anti-S1 titers were lower in PAD. All subjects with PAD demonstrated restricted anti-S1 IgG antibody repertoires, with usage of <5 IGHV subfamilies (median: 3; range 2-4), compared to ≥5 for the 11 HC subjects (P < .001). IGHV3-7 utilization was far less common in patients with PAD than in HCs (2 of 12 vs 10 of 11; P = .001). Amino acid substitutions due to somatic hypermutation per subfamily did not differ between groups. Anti-S1 IgM was present in 64% and 50% of HC and PAD cohorts, respectively, and did not differ significantly between HCs and patients with PAD. CONCLUSIONS: This study demonstrates the breadth of anti-S1 antibodies elicited by vaccination at the proteome level and identifies stereotypical restriction of IGHV utilization in the IgG repertoire in patients with PAD compared with HC subjects. Despite uniformly pauci-clonal antibody repertoires some patients with PAD generated potent serological responses, highlighting a possible limitation of traditional serological techniques. These findings suggest that IgG repertoire restriction is a key feature of antibody repertoires in PAD.
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COVID-19 , Enfermedades de Inmunodeficiencia Primaria , Humanos , Sustitución de Aminoácidos , Bioensayo , Vacunación , Inmunoglobulina G , Inmunoglobulina M , Anticuerpos AntiviralesRESUMEN
Although ACE2 is the primary receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here, we use whole-genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe Coronavirus Disease 2019 (COVID-19) infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , COVID-19/genética , Antivirales/farmacología , Enzima Convertidora de Angiotensina 2/metabolismo , Fibroblastos/metabolismo , Unión Proteica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
For over two decades, nanomaterials have been employed to facilitate intracellular delivery of small interfering RNA (siRNA), both in vitro and in vivo, to induce post-transcriptional gene silencing (PTGS) via RNA interference. Besides PTGS, siRNAs are also capable of transcriptional gene silencing (TGS) or epigenetic silencing, which targets the gene promoter in the nucleus and prevents transcription via repressive epigenetic modifications. However, silencing efficiency is hampered by poor intracellular and nuclear delivery. Here, polyarginine-terminated multilayered particles are reported as a versatile system for the delivery of TGS-inducing siRNA to potently suppress virus transcription in HIV-infected cells. siRNA is complexed with multilayered particles formed by layer-by-layer assembly of poly(styrenesulfonate) and poly(arginine) and incubated with HIV-infected cell types, including primary cells. Using deconvolution microscopy, uptake of fluorescently labeled siRNA is observed in the nuclei of HIV-1 infected cells. Viral RNA and protein are measured to confirm functional virus silencing from siRNA delivered using particles 16 days post-treatment. This work extends conventional particle-enabled PTGS siRNA delivery to the TGS pathway and paves the way for future studies on particle-delivered siRNA for efficient TGS of various diseases and infections, including HIV.
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Infecciones por VIH , VIH-1 , Humanos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , VIH-1/genética , VIH-1/metabolismo , Silenciador del Gen , Interferencia de ARN , Epigénesis Genética/genética , Infecciones por VIH/genética , Infecciones por VIH/terapiaRESUMEN
Patients with indolent lymphoma undertaking recurrent or continuous B cell suppression are at risk of severe COVID-19. Patients and healthy controls (HC; N = 13) received two doses of BNT162b2: follicular lymphoma (FL; N = 35) who were treatment naïve (TN; N = 11) or received immunochemotherapy (ICT; N = 23) and Waldenström's macroglobulinemia (WM; N = 37) including TN (N = 9), ICT (N = 14), or treated with Bruton's tyrosine kinase inhibitors (BTKi; N = 12). Anti-spike immunoglobulin G (IgG) was determined by a high-sensitivity flow-cytometric assay, in addition to live-virus neutralization. Antigen-specific T cells were identified by coexpression of CD69/CD137 and CD25/CD134 on T cells. A subgroup (N = 29) were assessed for third mRNA vaccine response, including omicron neutralization. One month after second BNT162b2, median anti-spike IgG mean fluorescence intensity (MFI) in FL ICT patients (9977) was 25-fold lower than TN (245 898) and HC (228 255, p = .0002 for both). Anti-spike IgG correlated with lymphocyte count (r = .63; p = .002), and time from treatment (r = .56; p = .007), on univariate analysis, but only with lymphocyte count on multivariate analysis (p = .03). In the WM cohort, median anti-spike IgG MFI in BTKi patients (39 039) was reduced compared to TN (220 645, p = .0008) and HC (p < .0001). Anti-spike IgG correlated with neutralization of the delta variant (r = .62, p < .0001). Median neutralization titer for WM BTKi (0) was lower than HC (40, p < .0001) for early-clade and delta. All cohorts had functional T cell responses. Median anti-spike IgG decreased 4-fold from second to third dose (p = .004). Only 5 of 29 poor initial responders assessed after third vaccination demonstrated seroconversion and improvement in neutralization activity, including to the omicron variant.
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COVID-19 , Linfoma no Hodgkin , Humanos , Inmunoglobulina G , SARS-CoV-2 , Vacuna BNT162 , COVID-19/prevención & control , Linfocitos T , Anticuerpos Antivirales , Anticuerpos Neutralizantes , VacunaciónRESUMEN
Purpose: The purpose of this study was to evaluate face mask-associated factors causing dry eye among medical students. Methodology: This was a cross-sectional study conducted on undergraduate medical and dental students, of all phases, while they were attending offline classes and were required to wear face masks in accordance with the government regulations. Sociodemographic data, ocular and medical history, face mask-wearing practices, screen usage, and quantification of symptoms using the modified Ocular Surface Disease Index (OSDI) questionnaire were collected. Objective tests were conducted in students having dry eye. The association of quantitative variables was done using ANOVA, Mann-Whitney, and Kruskal-Wallis test, whereas the Chi-square test was done for qualitative variables. Multivariate logistic regression was used to identify the risk factors for varying severity of dry eye. Results: The mean age of the 410 students was 21 ± 1.6 years. According to the OSDI, 39.51% (162/410) of students had dry eyes, 23.41% (96/410) had mild dry eye, 8.78% (36/410) had moderate dry eye, and 7.32% (30/410) had severe dry eye. Face mask-associated factors which were significantly linked to dry eye were N95 masks, loose-fit masks, and 6-8 h of continuous mask use. The Schirmer's test and tear film break-up time were performed on 29 and 20 students, respectively, mean values being 19.25 ± 5.29 mm and 10.15 ± 1.41 s for nonsevere and 6.53 ± 1.55 mm and 5.3 ± 0.98 s for severe dry eye, respectively. Conclusion: It is important to educate medical students and create awareness regarding "face mask-appropriate behavior" to reduce the chances of dry eye secondary to face masks use.
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Background: Long-term immunity to SARS-CoV-2 infection, including neutralizing antibodies and T cell-mediated immunity, is required in a very large majority of the population in order to reduce ongoing disease burden. Methods: We have investigated the association between memory CD4 and CD8 T cells and levels of neutralizing antibodies in convalescent COVID-19 subjects. Findings: Higher titres of convalescent neutralizing antibodies were associated with significantly higher levels of RBD-specific CD4 T cells, including specific memory cells that proliferated vigorously in vitro. Conversely, up to half of convalescent individuals had low neutralizing antibody titres together with a lack of receptor binding domain (RBD)-specific memory CD4 T cells. These low antibody subjects had other, non-RBD, spike-specific CD4 T cells, but with more of an inhibitory Foxp3+ and CTLA-4+ cell phenotype, in contrast to the effector T-bet+, cytotoxic granzymes+ and perforin+ cells seen in RBD-specific memory CD4 T cells from high antibody subjects. Single cell transcriptomics of antigen-specific CD4+ T cells from high antibody subjects similarly revealed heterogenous RBD-specific CD4+ T cells that comprised central memory, transitional memory and Tregs, as well as cytotoxic clusters containing diverse TCR repertoires, in individuals with high antibody levels. However, vaccination of low antibody convalescent individuals led to a slight but significant improvement in RBD-specific memory CD4 T cells and increased neutralizing antibody titres. Interpretation: Our results suggest that targeting CD4 T cell epitopes proximal to and within the RBD-region should be prioritized in booster vaccines.