Structure-guided T cell vaccine design for SARS-CoV-2 variants and sarbecoviruses.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that escape convalescent and vaccine-induced antibody responses has renewed focus on the development of broadly protective T-cell-based vaccines. Here, we apply structure-based network analysis and assessments of HLA class I peptide stability to define mutationally constrained CD8+ T cell epitopes across the SARS-CoV-2 proteome. Highly networked residues are conserved temporally among circulating variants and sarbecoviruses and disproportionately impair spike pseudotyped lentivirus infectivity when mutated. Evaluation of HLA class I stabilizing activity for 18 globally prevalent alleles identifies CD8+ T cell epitopes within highly networked regions with limited mutational frequencies in circulating SARS-CoV-2 variants and deep-sequenced primary isolates. Moreover, these epitopes elicit demonstrable CD8+ T cell reactivity in convalescent individuals but reduced recognition in recipients of mRNA-based vaccines. These data thereby elucidate key mutationally constrained regions and immunogenic epitopes in the SARS-CoV-2 proteome for a global T-cell-based vaccine against emerging variants and SARS-like coronaviruses.

Site of Origin of the Ophthalmic Artery Influences the Risk for Retinal Versus Cerebral Embolic Events.

BACKGROUND: Embolic events leading to retinal ischemia or cerebral ischemia share common risk factors; however, it has been well documented that the rate of concurrent cerebral infarction is higher in patients with a history of transient ischemic attack (TIA) than in those with monocular vision loss (MVL) due to retinal ischemia. Despite the fact that emboli to the ophthalmic artery (OA) and middle cerebral artery share the internal carotid artery (ICA) as a common origin or transit for emboli, the asymmetry in their final destination has not been fully explained. We hypothesize that the anatomic location of the OA takeoff from the ICA may contribute to the differential flow of small emboli to the retinal circulation vs the cerebral circulation. METHODS: We report a retrospective, comparative, case-control study on 28 patients with retinal ischemia and 26 patients with TIA or cerebral infarction caused by embolic events. All subjects underwent either computed tomography angiography or MRA. The location of the ipsilateral OA origin off the ICA was then graded in a blinded fashion and compared between cohorts. Vascular risk factors were collected for all patients, including age, sex, hypertension, hyperlipidemia, arrhythmia, diabetes, coronary artery disease, and smoking. RESULTS: We find that in patients with retinal ischemia of embolic etiology, the ipsilateral OA takeoff from the ICA is more proximal than in patients with cerebral infarcts or TIA (P = 0.0002). We found no statistically significant differences in demographic, vascular, or systemic risk factors. CONCLUSIONS: We find that the mean anatomical location of the OA takeoff from the ICA is significantly more proximal in patients with MVL due to retinal ischemia compared with patients with TIA or cerebral ischemia. This finding contributes significantly to our understanding of a long observed but poorly understood phenomenon that patients with MVL are less likely to have concurrent cerebral ischemia than are patients with TIA.

Annotation of loci from genome-wide association studies using tissue-specific quantitative interaction proteomics.

Genome-wide association studies (GWAS) have identified thousands of loci associated with complex traits, but it is challenging to pinpoint causal genes in these loci and to exploit subtle association signals. We used tissue-specific quantitative interaction proteomics to map a network of five genes involved in the Mendelian disorder long QT syndrome (LQTS). We integrated the LQTS network with GWAS loci from the corresponding common complex trait, QT-interval variation, to identify candidate genes that were subsequently confirmed in Xenopus laevis oocytes and zebrafish. We used the LQTS protein network to filter weak GWAS signals by identifying single-nucleotide polymorphisms (SNPs) in proximity to genes in the network supported by strong proteomic evidence. Three SNPs passing this filter reached genome-wide significance after replication genotyping. Overall, we present a general strategy to propose candidates in GWAS loci for functional studies and to systematically filter subtle association signals using tissue-specific quantitative interaction proteomics.

Human genetics in rheumatoid arthritis guides a high-throughput drug screen of the CD40 signaling pathway.

Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10(-9)). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10(-9)), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA-approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA.

Proteins encoded in genomic regions associated with immune-mediated disease physically interact and suggest underlying biology.

Genome-wide association studies (GWAS) have defined over 150 genomic regions unequivocally containing variation predisposing to immune-mediated disease. Inferring disease biology from these observations, however, hinges on our ability to discover the molecular processes being perturbed by these risk variants. It has previously been observed that different genes harboring causal mutations for the same Mendelian disease often physically interact. We sought to evaluate the degree to which this is true of genes within strongly associated loci in complex disease. Using sets of loci defined in rheumatoid arthritis (RA) and Crohn's disease (CD) GWAS, we build protein-protein interaction (PPI) networks for genes within associated loci and find abundant physical interactions between protein products of associated genes. We apply multiple permutation approaches to show that these networks are more densely connected than chance expectation. To confirm biological relevance, we show that the components of the networks tend to be expressed in similar tissues relevant to the phenotypes in question, suggesting the network indicates common underlying processes perturbed by risk loci. Furthermore, we show that the RA and CD networks have predictive power by demonstrating that proteins in these networks, not encoded in the confirmed list of disease associated loci, are significantly enriched for association to the phenotypes in question in extended GWAS analysis. Finally, we test our method in 3 non-immune traits to assess its applicability to complex traits in general. We find that genes in loci associated to height and lipid levels assemble into significantly connected networks but did not detect excess connectivity among Type 2 Diabetes (T2D) loci beyond chance. Taken together, our results constitute evidence that, for many of the complex diseases studied here, common genetic associations implicate regions encoding proteins that physically interact in a preferential manner, in line with observations in Mendelian disease.

Trends in Anti-Vascular Endothelial Growth Factor Original Medicare Part B Claims in the United States, 2014-2019.

PURPOSE: To characterize trends in use of and expenditure for the intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) agents aflibercept, ranibizumab, and bevacizumab among the population enrolled in Original Medicare from 2014 to 2019. METHODS: The Centers for Medicare and Medicaid Services Physician and Other Supplier Public Use File was used to extract Medicare Part B fee-for-service outpatient injection claims data submitted by ophthalmologists. Multivariable linear regression models were used to evaluate the association between reimbursement, ophthalmologist availability, and agent administration rate. RESULTS: Between 2014 and 2019, 17,588,995 intravitreal injection claims were filed by 4218 US ophthalmologists. Medicare costs for anti-VEGF injections increased from 2.51 B USD in 2014 to 4.02 B USD in 2019. Increased state-level ophthalmologist availability and incremental increases in average reimbursement amounts were found to be significantly associated with a 6.8-fold variation in 2019 overall anti-VEGF injection rates across states. CONCLUSIONS: Medicare injection rates and costs for anti-VEGF injections have both increased between 2014 and 2019, largely driven by increased aflibercept use. There is a significant association between ophthalmologist availability and anti-VEGF injection rate on the state level, suggesting access to care may contribute to the observed state-level disparities in intravitreal injection rates. Further characterization of factors contributing to the state-level variation in injection rates of individual anti-VEGF agents may help inform interventions promoting equitable access to and use of these drugs.

Structure-based network analysis predicts pathogenic variants in human proteins associated with inherited retinal disease.

Advances in gene sequencing technologies have accelerated the identification of genetic variants, but better tools are needed to understand which are causal of disease. This would be particularly useful in fields where gene therapy is a potential therapeutic modality for a disease-causing variant such as inherited retinal disease (IRD). Here, we apply structure-based network analysis (SBNA), which has been successfully utilized to identify variant-constrained amino acid residues in viral proteins, to identify residues that may cause IRD if subject to missense mutation. SBNA is based entirely on structural first principles and is not fit to specific outcome data, which makes it distinct from other contemporary missense prediction tools. In 4 well-studied human disease-associated proteins (BRCA1, HRAS, PTEN, and ERK2) with high-quality structural data, we find that SBNA scores correlate strongly with deep mutagenesis data. When applied to 47 IRD genes with available high-quality crystal structure data, SBNA scores reliably identified disease-causing variants according to phenotype definitions from the ClinVar database. Finally, we applied this approach to 63 patients at Massachusetts Eye and Ear (MEE) with IRD but for whom no genetic cause had been identified. Untrained models built using SBNA scores and BLOSUM62 scores for IRD-associated genes successfully predicted the pathogenicity of novel variants (AUC = 0.851), allowing us to identify likely causative disease variants in 40 IRD patients. Model performance was further augmented by incorporating orthogonal data from EVE scores (AUC = 0.927), which are based on evolutionary multiple sequence alignments. In conclusion, SBNA can used to successfully identify variants as causal of disease in human proteins and may help predict variants causative of IRD in an unbiased fashion.

Rare genetic variation in VE-PTP is associated with central serous chorioretinopathy, venous dysfunction and glaucoma.

Central serous chorioretinopathy (CSC) is a fluid maculopathy whose etiology is not well understood. Abnormal choroidal veins in CSC patients have been shown to have similarities with varicose veins. To identify potential mechanisms, we analyzed genotype data from 1,477 CSC patients and 455,449 controls in FinnGen. We identified an association for a low-frequency (AF=0.5%) missense variant (rs113791087) in the gene encoding vascular endothelial protein tyrosine phosphatase (VE-PTP) (OR=2.85, P=4.5×10-9). This was confirmed in a meta-analysis of 2,452 CSC patients and 865,767 controls from 4 studies (OR=3.06, P=7.4×10-15). Rs113791087 was associated with a 56% higher prevalence of retinal abnormalities (35.3% vs 22.6%, P=8.0×10-4) in 708 UK Biobank participants and, surprisingly, with varicose veins (OR=1.31, P=2.3×10-11) and glaucoma (OR=0.82, P=6.9×10-9). Predicted loss-of-function variants in VEPTP, though rare in number, were associated with CSC in All of Us (OR=17.10, P=0.018). These findings highlight the significance of VE-PTP in diverse ocular and systemic vascular diseases.

Effectiveness of a telemedicine program for triage and diagnosis of emergent ophthalmic conditions.

BACKGROUND: To study the utility of a teleophthalmology program to diagnose and triage common ophthalmic complaints presenting to an ophthalmic emergency room. METHODS: Prospective, observational study of 258 eyes of 129 patients presenting to the Massachusetts Eye and Ear Infirmary Emergency Ward (MEE EW) who completed a questionnaire to gather chief complaint (CC), history of present illness, and medical history. Anterior and posterior segment photographs were collected via iPhone 5 C camera and a Canon non-mydriatic fundus camera, respectively. Ophthalmic vital signs were collected. All information was reviewed remotely by three ophthalmologists; a diagnosis and urgency designation were recorded. The remote assessment was compared to gold standard in-person assessment. RESULTS: The 129 recruited patients collectively contributed 220 visual complaints, of which 121 (55%) were from females with mean age 56.5 years (range 24-89). Sensitivities and specificities for telemedical triage were as follows: eye pain (n = 56; sensitivity: 0.58, CI [0.41, 0.74]; specificity: 0.91, CI [0.80, 1]), eye redness (n = 54; 0.68, CI [0.50, 0.86]; 0.93, CI [0.84, 1]), blurry vision (n = 68; 0.73, CI [0.60, 0.86]; 0.91, CI [0.80, 1]), and eyelid complaints (n = 42; 0.67, CI [0.43, 0.91]; 0.96, CI [0.89, 1]). The remote diagnostic accuracies, as stratified by CC, were eye pain (27/56; 48.21%), eye redness: (32/54; 59.26%), blurry vision: (30/68; 44.11%), eyelid (24/42; 57.14%). CONCLUSIONS: Telemedical examination of emergent ophthalmic complaints consisting of a patient questionnaire, anterior segment and fundus photos, and ophthalmic vital signs, may be useful to reliably triage eye disease based on presenting complaint.

Open Globe Injuries From Garage Door Springs.

We describe seven patients who were attempting to repair their garage door when a spring dislodged at high velocity, resulting in open globe injury. All patients were seen at Massachusetts Eye and Ear between the years 2008 and 2023. Their final visual acuities ranged from 20/125 to no light perception. Open globe injury appears to be a risk of attempts to repair a garage door by people who are inexperienced in doing so. [Ophthalmic Surg Lasers Imaging Retina 2023;54:666-669.].

Structure-based network analysis predicts mutations associated with inherited retinal disease.

With continued advances in gene sequencing technologies comes the need to develop better tools to understand which mutations cause disease. Here we validate structure-based network analysis (SBNA)1,2 in well-studied human proteins and report results of using SBNA to identify critical amino acids that may cause retinal disease if subject to missense mutation. We computed SBNA scores for genes with high-quality structural data, starting with validating the method using 4 well-studied human disease-associated proteins. We then analyzed 47 inherited retinal disease (IRD) genes. We compared SBNA scores to phenotype data from the ClinVar database and found a significant difference between benign and pathogenic mutations with respect to network score. Finally, we applied this approach to 65 patients at Massachusetts Eye and Ear (MEE) who were diagnosed with IRD but for whom no genetic cause was found. Multivariable logistic regression models built using SBNA scores for IRD-associated genes successfully predicted pathogenicity of novel mutations, allowing us to identify likely causative disease variants in 37 patients with IRD from our clinic. In conclusion, SBNA can be meaningfully applied to human proteins and may help predict mutations causative of IRD.

Overlap of Genetic Loci for Central Serous Chorioretinopathy With Age-Related Macular Degeneration.

Importance: Central serous chorioretinopathy (CSC) is a serous maculopathy of unknown etiology. Two of 3 previously reported CSC genetic risk loci are also associated with AMD. Improved understanding of CSC genetics may broaden our understanding of this genetic overlap and unveil mechanisms in both diseases. Objective: To identify novel genetic risk factors for CSC and compare genetic risk factors for CSC and AMD. Design, Setting, and Participants: Using International Classification of Diseases, Ninth (ICD-9) and Tenth (ICD-10) Revision code-based inclusion and exclusion criteria, patients with CSC and controls were identified in both the FinnGen study and the Estonian Biobank (EstBB). Also included in a meta-analysis were previously reported patients with chronic CSC and controls. Data were analyzed from March 1 to September 31, 2022. Main Outcomes and Measures: Genome-wide association studies (GWASs) were performed in the biobank-based cohorts followed by a meta-analysis of all cohorts. The expression of genes prioritized by the polygenic priority score and nearest-gene methods were assessed in cultured choroidal endothelial cells and public ocular single-cell RNA sequencing data sets. The predictive utility of polygenic scores (PGSs) for CSC and AMD were evaluated in the FinnGen study. Results: A total of 1176 patients with CSC and 526 787 controls (312 162 female [59.3%]) were included in this analysis: 552 patients with CSC and 343 461 controls were identified in the FinnGen study, 103 patients with CSC and 178 573 controls were identified in the EstBB, and 521 patients with chronic CSC and 3577 controls were included in a meta-analysis. Two previously reported CSC risk loci were replicated (near CFH and GATA5) and 3 novel loci were identified (near CD34/46, NOTCH4, and PREX1). The CFH and NOTCH4 loci were associated with AMD but in the opposite direction. Prioritized genes showed increased expression in cultured choroidal endothelial cells compared with other genes in the loci (median [IQR] of log 2 [counts per million], 7.3 [0.6] vs 4.7 [3.7]; P = .004) and were differentially expressed in choroidal vascular endothelial cells in single-cell RNA sequencing data (mean [SD] fold change, 2.05 [0.38] compared with other cell types; P < 7.1 × 10-20). A PGS for AMD was predictive of reduced CSC risk (odds ratio, 0.76; 95% CI, 0.70-0.83 per +1 SD in AMD-PGS; P = 7.4 × 10-10). This association may have been mediated by loci containing complement genes. Conclusions and Relevance: In this 3-cohort genetic association study, 5 genetic risk loci for CSC were identified, highlighting a likely role for genes involved in choroidal vascular function and complement regulation. Results suggest that polygenic AMD risk was associated with reduced risk of CSC and that this genetic overlap was largely due to loci containing complement genes.

Identifying relationships among genomic disease regions: predicting genes at pathogenic SNP associations and rare deletions.

Translating a set of disease regions into insight about pathogenic mechanisms requires not only the ability to identify the key disease genes within them, but also the biological relationships among those key genes. Here we describe a statistical method, Gene Relationships Among Implicated Loci (GRAIL), that takes a list of disease regions and automatically assesses the degree of relatedness of implicated genes using 250,000 PubMed abstracts. We first evaluated GRAIL by assessing its ability to identify subsets of highly related genes in common pathways from validated lipid and height SNP associations from recent genome-wide studies. We then tested GRAIL, by assessing its ability to separate true disease regions from many false positive disease regions in two separate practical applications in human genetics. First, we took 74 nominally associated Crohn's disease SNPs and applied GRAIL to identify a subset of 13 SNPs with highly related genes. Of these, ten convincingly validated in follow-up genotyping; genotyping results for the remaining three were inconclusive. Next, we applied GRAIL to 165 rare deletion events seen in schizophrenia cases (less than one-third of which are contributing to disease risk). We demonstrate that GRAIL is able to identify a subset of 16 deletions containing highly related genes; many of these genes are expressed in the central nervous system and play a role in neuronal synapses. GRAIL offers a statistically robust approach to identifying functionally related genes from across multiple disease regions--that likely represent key disease pathways. An online version of this method is available for public use (http://www.broad.mit.edu/mpg/grail/).

Bacillary Layer Detachment Associated With Endogenous Fungal Endophthalmitis.

Purpose: To recognize the novel finding of a bacillary layer detachment (BALAD) secondary to endogenous fungal endophthalmitis. Methods: Chart review, literature review. Results: BALAD is a recently described condition in which the photoreceptor layer splits at the level of the inner segment myoid. We describe a case of BALAD associated with endogenous fungal endophthalmitis and subsequent development of choroidal neovascularization, although it is unclear if BALAD contributed to neovessel formation. Conclusions: BALAD is generally seen in the setting of inflammatory or infectious retinal diseases. This is the first report of BALAD secondary to endogenous fungal endophthalmitis.

Two Cases of Cutibacterium acnes (C acnes) Endophthalmitis Manifesting With Unusual Epiretinal Deposits.

This article describes two cases of delayed-onset Cutibacterium acnes (C acnes) endophthalmitis 1 month after cataract surgery manifesting with unusual epiretinal deposits. Both patients were referred for persistent inflammation after cataract surgery. After failing to respond to a vitreous tap with injection of antibiotics, the patients underwent pars plana vitrectomy and in one of the cases partial posterior capsulectomy for a posterior capsular plaque. Intraoperatively, both cases were found to have unusual multifocal epiretinal deposits. The clinical presentations described here represent a highly unique manifestation of C acnes endophthalmitis distinct from the classic anterior segment findings. [Ophthalmic Surg Lasers Imaging Retina. 2022;53:164-167.].

Hypopyon and White Cataract Due to an Intraocular Helminth.

We describe a case of a 29-year-old man with a history of intravenous drug use and vague history of eye trauma who presented with a hypopyon and white cataract in the right eye. He underwent pars plana vitrectomy and lensectomy; his anterior chamber aspirate revealed a single helminth on calcofluor stain. We suspect that his helminth infection may be secondary to unsanitary eating and drinking practices. As overall hygiene and dietary habits have improved during the years, parasitic helminth infections are relatively rare in nonendemic areas, especially in the nonpediatric population. [Ophthalmic Surg Lasers Imaging Retina. 2022;53:168-171.].

SARS-CoV-2 RNA Detected in Vitreous Samples Obtained at Autopsy.

Purpose: This work aims to examine the vitreous of autopsy patients with COVID-19 for the presence of SARS-CoV-2 RNA. Methods: Four deceased patients with COVID-19 had an autopsy at Massachusetts General Hospital. Two control specimens were obtained from patients undergoing retinal detachment repair with negative preoperative polymerase chain reaction (PCR) testing for SARS-CoV-2 RNA. Vitreous specimens were obtained from autopsy patients with COVID-19 after povidone was placed on the ocular surface to decrease the risk of contamination of the vitreous specimen. SARS-CoV-2 RNA for gene N (nucleocapsid) was tested using reverse transcription-PCR. Results: SARS-CoV-2 RNA was detected in the vitreous of 2 of 4 autopsy patients who died from complications of COVID-19. Conclusions: SARS-CoV-2 RNA can penetrate into the vitreous of systemically infected patients, which might present risks to operating room personnel during ophthalmic surgical procedures.

Single-cell RNA sequencing: An overview for the ophthalmologist.

Understanding the molecular composition of pathogenic tissues is a critical step in understanding the pathophysiology of disease and designing therapeutics. First described in 2009, single cell RNA sequencing (scRNAseq) is a methodology whereby thousands of cells are simultaneously isolated into individual micro-environments that can be altered experimentally and the genome-wide RNA expression of each cell is captured. It has undergone significant technological improvement over the last decade and gained tremendous popularity. scRNAseq is an improvement over prior pooled RNA analyses which cannot identify the cellular composition and heterogeneity of a tissue of interest. This new approach offers new opportunity for new discovery, as tissue samples can now be sub-categorized into groups of cell types based on genome-wide gene expression in an unbiased fashion. As ophthalmologists, we are uniquely positioned to obtain pathologic samples from the eye for further study. ScRNAseq has already been applied in ophthalmology to characterize retinal tissue, and it may offer the key to understanding various pathological processes in the future.

Aflibercept for Retinopathy of Prematurity: A Systematic Review and Meta-Analysis.

BACKGROUND AND OBJECTIVE: To determine the effectiveness of aflibercept in retinopathy of prematurity (ROP). PATIENTS AND METHODS: We performed a systematic review and meta-analysis of proportions from the literature in PubMed and Cochrane Library using search terms related to the use of aflibercept in ROP. Studies in non-preterm infants or that did not use aflibercept as the initial treatment were excluded. Risk of bias was assessed by the ROBINS-I (Risk Of Bias in Non-randomized Studies of Interventions) tool. RESULTS: We identified six case series. Collectively, 218 eyes were treated with aflibercept for ROP. We found an average 97% (95% confidence interval [CI], 93% to 99%) regression rate with aflibercept and an average 16% (95% CI, 5% to 41%) recurrence rate. With the exception of one outlier study, these numbers are similar to previous reports using anti-vascular endothelial growth factor (VEGF) agents in ROP. CONCLUSIONS: Aflibercept holds promise for use in ROP and has been demonstrated to be efficacious in six case series. Randomized, controlled clinical trials appear warranted to compare aflibercept with other anti-VEGF agents. [Ophthalmic Surg Lasers Imaging. 2021;52:673-681.].