Role of IgG avidity in eyes with active Toxoplasma retinochoroiditis.

PURPOSE: To study the role of Toxoplasma IgG avidity in evaluating the stage of systemic infection during manifestation as toxoplasma retinochoroiditis and its clinical implications in eastern India. METHODS: Retrospective chart review of Toxoplasma retinochoroiditis cases with Toxoplasma serology for IgG, IgM, and IgG avidity. RESULTS: Included in this study were 17 eyes of 17 patients who had active retinitis located in the macula (14), mid-periphery (2), or periphery (1). They were either primary lesions (12) or reactivations (5). All the cases had Toxoplasma IgG positive; one case had IgM positivity, while all the cases had high IgG avidity values. IgG avidity had a positive correlation with the duration of symptoms. CONCLUSION: We observed high IgG avidity values in active retinochoroiditis in both primary ocular Toxoplasmosis and reactivation subgroups. These results indicate a late ocular manifestation after initial systemic infection with a possible incubation period ranging from 5 weeks to 5 months.

Macular Punctate Lesions Presenting as a Primary Manifestation of Ocular Toxoplasmosis.

PURPOSE: To study clinical features and outcomes of primary ocular Toxoplasmosis (OT) cases presenting as macular punctate lesions. METHODS: Retrospective review of three cases of OT with positive Toxoplasma serology. RESULTS: We describe three cases presenting as primary OT with no evidence of old retinochoroidal scar in either eye. All the cases had multiple foveal or extrafoveal, punctate, inner/outer, or combined lesions at macula with minimal vitreous reaction. During the first/primary episode, all the lesions resolved with 1. retinal atrophy, thinning (n = 1) or 2. Progressed to limited full-thickness retinitis lesions (n = 2). Recurrence as typical retinochoroiditis was seen in one eye. More than four-fold IgG positivity was seen in all cases while IgM positivity was seen in two cases. CONCLUSIONS: Macular punctate lesions (inner/outer/combination) can be the primary manifestation of ocular toxoplasmosis in the absence of old retinochoroiditis scars in either eye.

Rapid, high throughput, automated detection of SARS-CoV-2 neutralizing antibodies against Wuhan-WT, delta and omicron BA1, BA2 spike trimers.

Traditional cellular and live-virus methods for detection of SARS-CoV-2 neutralizing antibodies (nAbs) are labor- and time-intensive, and thus not suited for routine use in the clinical lab to predict vaccine efficacy and natural immune protection. Here, we report the development and validation of a rapid, high throughput method for measuring SARS-CoV-2 nAbs against native-like trimeric spike proteins. This assay uses a blockade of human angiotensin converting enzyme 2 (hACE-2) binding (BoAb) approach in an automated digital immunoassay on the Quanterix HD-X platform. BoAb assays using Wuhan-WT (vaccine strain), delta (B.1.167.2), omicron BA1 and BA2 variant viral strains showed strong correlation with cell-based pseudovirus neutralization activity (PNA) and live-virus neutralization activity. Importantly, we were able to detect similar patterns of delta and omicron variant resistance to neutralization in samples with paired vaccine strain and delta variant BoAb measurements. Finally, we screened clinical samples from patients with or without evidence of SARS-CoV-2 exposure by a single-dilution screening version of our assays, finding significant nAb activity only in exposed individuals. Importantly, this completely automated assay can be performed in 4 h to measure neutralizing antibody titers for 16 samples over 8 serial dilutions or, 128 samples at a single dilution with replicates. In principle, these assays offer a rapid, robust, and scalable alternative to time-, skill-, and cost-intensive standard methods for measuring SARS-CoV-2 nAb levels.

Light chain of a public SARS-CoV-2 class-3 antibody modulates neutralization against Omicron.

The pairing of antibody genes IGHV2-5/IGLV2-14 is established as a public immune response that potently cross-neutralizes SARS-CoV-2 variants, including Omicron, by targeting class-3/RBD-5 epitopes in the receptor binding domain (RBD). LY-CoV1404 (bebtelovimab) exemplifies this, displaying exceptional potency against Omicron sub-variants up to BA.5. Here, we report a human antibody, 002-S21B10, encoded by the public clonotype IGHV2-5/IGLV2-14. While 002-S21B10 neutralized key SARS-CoV-2 variants, it did not neutralize Omicron, despite sharing >92% sequence similarity with LY-CoV1404. The structure of 002-S21B10 in complex with spike trimer plus structural and sequence comparisons with LY-CoV1404 and other IGHV2-5/IGLV2-14 antibodies revealed significant variations in light-chain orientation, paratope residues, and epitope-paratope interactions that enable some antibodies to neutralize Omicron but not others. Confirming this, replacing the light chain of 002-S21B10 with the light chain of LY-CoV1404 restored 002-S21B10's binding to Omicron. Understanding such Omicron evasion from public response is vital for guiding therapeutics and vaccine design.

Molecular basis of SARS-CoV-2 Omicron variant evasion from shared neutralizing antibody response.

Understanding the molecular features of neutralizing epitopes is important for developing vaccines/therapeutics against emerging SARS-CoV-2 variants. We describe three monoclonal antibodies (mAbs) generated from COVID-19 recovered individuals during the first wave of the pandemic in India. These mAbs had publicly shared near germline gene usage and potently neutralized Alpha and Delta, poorly neutralized Beta, and failed to neutralize Omicron BA.1 SARS-CoV-2 variants. Structural analysis of these mAbs in complex with trimeric spike protein showed that all three mAbs bivalently bind spike with two mAbs targeting class 1 and one targeting a class 4 receptor binding domain epitope. The immunogenetic makeup, structure, and function of these mAbs revealed specific molecular interactions associated with the potent multi-variant binding/neutralization efficacy. This knowledge shows how mutational combinations can affect the binding or neutralization of an antibody, which in turn relates to the efficacy of immune responses to emerging SARS-CoV-2 escape variants.

A comparative randomized controlled study of modified 4 in 1 block versus IPACK plus adductor canal block for post-operative analgesia in total knee arthroplasty.

Background and Aims: Postoperative analgesia for Total Knee Arthroplasty (TKA) is paramount for early mobilisation and rehabilitation. The newer motor sparing peripheral nerve blocks for analgesia for TKA are 4 in 1 block, modified 4 in 1 block, infiltration between popliteal artery and capsule of the knee (IPACK) block along with adductor canal block (ACB). We hypothesised that Modified 4 in 1 block is as efficient as the already proven technique of combined IPACK and ACB in providing post-operative analgesia to the patients of TKA. Methods: Seventy patients fulfilling the inclusion criteria posted for TKA surgery were randomised into two groups: Modified 4 in 1 block group (Group - M) and combined IPACK + ACB group (Group - I). After thorough preoperative evaluation and with mimimum standard monitoring the patients received sub-arachnoid block followed by the desired peripheral nerve block as per the group. After the surgery the visual analog scale (VAS) pain score was compared at 3, 6, 12, 24 hours postoperatively and tabulated. Results: The mean pain scores between both the groups was comparable at 3 hours, 6 hours and 24 hours. But at 12 hours after the surgery, VAS was less in Group-M in comparison to Group-I, Haemodynamic parameters were comparable between both the groups. None of the patients in both the groups showed any complications like muscle weakness in the post-operative period. Conclusion: Modified 4 in 1 block is a new and novel technique for the TKA surgeries and is comparable with already established combined IPACK+ACB technique for providing adequate postoperative analgesia after TKA.

Beaded Pearls Appearance in Syphilitic Chorioretinitis.

PURPOSE: To report an atypical case of bilateral syphilitic chorioretinitis. METHODS: A case report. RESULTS: A young male presented with bilateral pigmentary retinal changes along with multifocal chorioretinal lesions along the blood vessels giving a "beaded pearl" appearance. He was a hitherto undiagnosed case of human immunodeficiency virus infection and was diagnosed to have syphilis. He had a favourable visual and anatomical outcome following treatment. CONCLUSION: Multifocal chorioretinal lesions along blood vessels forming a "beaded pearls" appearance can be a rare and unique presentation of syphilis.

Molecular basis of SARS-CoV-2 Omicron variant evasion from shared neutralizing antibody response.

A detailed understanding of the molecular features of the neutralizing epitopes developed by viral escape mutants is important for predicting and developing vaccines or therapeutic antibodies against continuously emerging SARS-CoV-2 variants. Here, we report three human monoclonal antibodies (mAbs) generated from COVID-19 recovered individuals during first wave of pandemic in India. These mAbs had publicly shared near germline gene usage and potently neutralized Alpha and Delta, but poorly neutralized Beta and completely failed to neutralize Omicron BA.1 SARS-CoV-2 variants. Structural analysis of these three mAbs in complex with trimeric spike protein showed that all three mAbs are involved in bivalent spike binding with two mAbs targeting class-1 and one targeting class-4 Receptor Binding Domain (RBD) epitope. Comparison of immunogenetic makeup, structure, and function of these three mAbs with our recently reported class-3 RBD binding mAb that potently neutralized all SARS-CoV-2 variants revealed precise antibody footprint, specific molecular interactions associated with the most potent multi-variant binding / neutralization efficacy. This knowledge has timely significance for understanding how a combination of certain mutations affect the binding or neutralization of an antibody and thus have implications for predicting structural features of emerging SARS-CoV-2 escape variants and to develop vaccines or therapeutic antibodies against these.

Structural insights for neutralization of Omicron variants BA.1, BA.2, BA.4, and BA.5 by a broadly neutralizing SARS-CoV-2 antibody.

In this study, by characterizing several human monoclonal antibodies (mAbs) isolated from single B cells of the COVID-19-recovered individuals in India who experienced ancestral Wuhan strain (WA.1) of SARS-CoV-2 during early stages of the pandemic, we found a receptor binding domain (RBD)-specific mAb 002-S21F2 that has rare gene usage and potently neutralized live viral isolates of SARS-CoV-2 variants including Alpha, Beta, Gamma, Delta, and Omicron sublineages (BA.1, BA.2, BA.2.12.1, BA.4, and BA.5) with IC50 ranging from 0.02 to 0.13 µg/ml. Structural studies of 002-S21F2 in complex with spike trimers of Omicron and WA.1 showed that it targets a conformationally conserved epitope on the outer face of RBD (class 3 surface) outside the ACE2-binding motif, thereby providing a mechanistic insights for its broad neutralization activity. The discovery of 002-S21F2 and the broadly neutralizing epitope it targets have timely implications for developing a broad range of therapeutic and vaccine interventions against SARS-CoV-2 variants including Omicron sublineages.

Deep mutational scanning identifies SARS-CoV-2 Nucleocapsid escape mutations of currently available rapid antigen tests.

The effects of mutations in continuously emerging variants of SARS-CoV-2 are a major concern for the performance of rapid antigen tests. To evaluate the impact of mutations on 17 antibodies used in 11 commercially available antigen tests with emergency use authorization, we measured antibody binding for all possible Nucleocapsid point mutations using a mammalian surface-display platform and deep mutational scanning. The results provide a complete map of the antibodies' epitopes and their susceptibility to mutational escape. Our data predict no vulnerabilities for detection of mutations found in variants of concern. We confirm this using the commercial tests and sequence-confirmed COVID-19 patient samples. The antibody escape mutational profiles generated here serve as a valuable resource for predicting the performance of rapid antigen tests against past, current, as well as any possible future variants of SARS-CoV-2, establishing the direct clinical and public health utility of our system.

Differential Modulation of Nuclear Receptor LRH-1 through Targeting Buried and Surface Regions of the Binding Pocket.

Liver receptor homologue-1 (LRH-1) is a phospholipid-sensing nuclear receptor that has shown promise as a target for alleviating intestinal inflammation and metabolic dysregulation in the liver. LRH-1 contains a large ligand-binding pocket, but generating synthetic modulators has been challenging. We have had recent success generating potent and efficacious agonists through two distinct strategies. We targeted residues deep within the pocket to enhance compound binding and residues at the mouth of the pocket to mimic interactions made by phospholipids. Here, we unite these two designs into one molecule to synthesize the most potent LRH-1 agonist to date. Through a combination of global transcriptomic, biochemical, and structural studies, we show that selective modulation can be driven through contacting deep versus surface polar regions in the pocket. While deep pocket contacts convey high affinity, contacts with the pocket mouth dominate allostery and provide a phospholipid-like transcriptional response in cultured cells.

The retinal vascular growth rate in babies with retinopathy of prematurity could indicate treatment need.

Purpose: To analyze the weekly rate of retinal vascular growth in treatment-naïve babies with various stages of retinopathy of prematurity (ROP) and validate if this could be a predictor of treatment need. Methods: Retrospective review of medical charts and retinal images of babies with various stages of ROP. The images were enhanced using red-green image enhancement software. Using the length of the horizontal disc diameter (DD) of each eye, the vessel growth was measured from the disc margin up to the vessel tip in fixed quadrants. The rate of vessel growth was the ratio of vessel length to the number of weeks it took to reach this length. The babies were divided into treatment warranting ROP (group 1), low-risk pre-threshold (type II) ROP (group 2,), and no-ROP (group 3) for analysis. The "no-ROP" group acted as normal control. Group 1 was further subdivided into 1A (threshold ROP), IB (aggressive posterior ROP), 1C (hybrid ROP), and ID (high-risk pre-threshold ROP). Results: Out of 436 eyes, groups 1, 2, and 3 had 238, 108, and 90 eyes, respectively. The mean rate of vascular outgrowth along with 95% confidence interval (CI) was 0.490 [0.487,0.520], 0.612 [0.599, 0.638], and 0.719 [0.703, 0.740] DD/week, respectively, for babies with "treatment warranting," "low risk pre-threshold" and "no ROP" groups, respectively. In our estimate, more than 80% of eyes with a vessel growth rate of 0.54 DD/week or less required treatment. Conclusion: A rate of retinal vascular growth less than 0.54 DD/week can be used to determine treatment requirements in babies with ROP.

A phospholipid mimetic targeting LRH-1 ameliorates colitis.

Phospholipids are ligands for nuclear hormone receptors (NRs) that regulate transcriptional programs relevant to normal physiology and disease. Here, we demonstrate that mimicking phospholipid-NR interactions is a robust strategy to improve agonists of liver receptor homolog-1 (LRH-1), a therapeutic target for colitis. Conventional LRH-1 modulators only partially occupy the binding pocket, leaving vacant a region important for phospholipid binding and allostery. Therefore, we constructed a set of molecules with elements of natural phospholipids appended to a synthetic LRH-1 agonist. We show that the phospholipid-mimicking groups interact with the targeted residues in crystal structures and improve binding affinity, LRH-1 transcriptional activity, and conformational changes at a key allosteric site. The best phospholipid mimetic markedly improves colonic histopathology and disease-related weight loss in a murine T cell transfer model of colitis. This evidence of in vivo efficacy for an LRH-1 modulator in colitis represents a leap forward in agonist development.

Rapid, high throughput, automated detection of SARS-CoV-2 neutralizing antibodies against native-like vaccine and delta variant spike trimers.

Traditional cellular and live-virus methods for detection of SARS-CoV-2 neutralizing antibodies (nAbs) are labor- and time-intensive, and thus not suited for routine use in the clinical lab to predict vaccine efficacy and natural immune protection. Here, we report the development and validation of a rapid, high throughput method for measuring SARS-CoV-2 nAbs against native-like trimeric spike proteins. This assay uses a blockade of hACE-2 binding (BoAb) approach in an automated digital immunoassay on the Quanterix HD-X platform. BoAb assays using vaccine and delta variant viral strains showed strong correlation with cell-based pseudovirus and live-virus neutralization activity. Importantly, we were able to detect similar patterns of delta variant resistance to neutralization in samples with paired vaccine and delta variant BoAb measurements. Finally, we screened clinical samples from patients with or without evidence of SARS-CoV-2 exposure by a single-dilution screening version of our assays, finding significant nAb activity only in exposed individuals. In principle, these assays offer a rapid, robust, and scalable alternative to time-, skill-, and cost-intensive standard methods for measuring SARS-CoV-2 nAb levels.

Rapid, high throughput, automated detection of SARS-CoV-2 neutralizing antibodies against native-like vaccine and delta variant spike trimers.

Traditional cellular and live-virus methods for detection of SARS-CoV-2 neutralizing antibodies (nAbs) are labor- and time-intensive, and thus not suited for routine use in the clinical lab to predict vaccine efficacy and natural immune protection. Here, we report the development and validation of a rapid, high throughput method for measuring SARS-CoV-2 nAbs against native-like trimeric spike proteins. This assay uses a blockade of hACE-2 binding (BoAb) approach in an automated digital immunoassay on the Quanterix HD-X platform. BoAb assays using vaccine and delta variant viral strains showed strong correlation with cell-based pseudovirus and live-virus neutralization activity. Importantly, we were able to detect similar patterns of delta variant resistance to neutralization in samples with paired vaccine and delta variant BoAb measurements. Finally, we screened clinical samples from patients with or without evidence of SARS-CoV-2 exposure by a single-dilution screening version of our assays, finding significant nAb activity only in exposed individuals. In principle, these assays offer a rapid, robust, and scalable alternative to time-, skill-, and cost-intensive standard methods for measuring SARS-CoV-2 nAb levels.

Posterior Subhyaloid Precipitates: 'KPs' of the Posterior Segment.

Purpose: To describe the course of posterior subhyaloid precipitates (PSPs) accompanying active retinitis. Methods: Retrospective study of three retinitis cases associated with PSPs and literature review. Results: Cases 1 and 2 were Toxoplasma retinochoroiditis. Case 3 had bilateral retinitis post-febrile illness. PSPs were yellowish-white spherical lesions seen in the presence of partial posterior hyaloid detachment (PVD) in the retro-hyaloid space lining the inferior PVD margin. In all 3 cases, PSPs resolved during treatment for primary disease. Resolution of PSPs preceded resolution of retinitis. Literature review revealed similar lesions in CMV retinitis and syphilis in addition to Toxoplasma retinochoroiditis. Conclusion: PSPs were seen in the presence of active retinitis and partial PVD when inflammatory aggregates gravitate inferiorly in the subhyaloid space. They were observed during active retinitis and their resolution preceded the resolution of retinitis, thus providing a potential marker for disease resolution.

Recent Advances in Diagnosis and Treatment of Infectious Uveitis Prevalent in Asia-Pacific Region.

ABSTRACT: The role of infections in intraocular inflammation is being increasingly recognized across the world. The Asia-Pacific region, being the single largest and most populous geographical entity on the planet, is home to a wide variety of such infections. Not surprisingly, there has been an explosion in the literature on infectious uveitis emerging from Asia-Pacific countries. In this review, we have covered recent advances in the diagnosis, treatment, and pathogenesis of common forms of infectious uveitis from the Asia-Pacific region. Much of the literature is focussed on the diagnosis of these infections by clinical criteria and laboratory investigations. There has also been an increased emphasis on the application of newer modes of ocular imaging and understanding pathomechanisms of ocular inflammation in these infections. Together this research has significantly improved our understanding of the diagnosis and management of infectious uveitis.